HoxD3, HoxA3, and HoxB3 compositions and methods for improved wound healing

ABSTRACT

The present invention provides methods and compositions useful in localized transfer of genetic material or proteins. Moreover, the present invention provides methods and compositions for improving and/or controlling wound healing by applying a wound care device comprising HoxD3 and/or HoxA3 and/or HoxB3. In addition, the present invention provides methods and compositions for improved wound healing in subjects having impaired healing capabilities, such as diabetic subjects.

This application is a Continuation-in-Part of U.S. Ser. No. 10/305,667,filed on Nov. 26, 2002, which is a Continuation-in-Part and claimsbenefit of international patent application PCT/US02/19020, filed onJun. 14, 2002, which claims benefit of provisional patent applicationU.S. Ser. No. 60/298,688, filed on Jun. 14, 2001, and provisional patentapplication U.S. Ser. No. 60/307,632, filed on Jul. 24, 2001.

The invention was made in part with Government support from the NationalInstitutes of Health, Grants K08 GM00674-01, P50 GM27345, and RO1CA85249. As such, the Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention provides methods and compositions useful inlocalized transfer of genetic material or proteins. Moreover, thepresent invention provides methods and compositions for improving and/orcontrolling wound healing by applying a wound care device comprisingHoxD3 and/or HoxA3 and/or HoxB3. In addition, the present inventionprovides methods and compositions for improved wound healing in subjectshaving impaired healing capabilities, such as diabetic subjects.

BACKGROUND OF THE INVENTION

Various methods are available for the transfer of genetic informationand proteins to cells. However, there remains a need in the art toprovide localized, high efficiency transfer of genetic information andproteins to cells and tissues, such that long-term benefits areprovided. In particular, methods and compositions are needed forsettings such as wound healing.

For example, during the process of healing, infection can occur. Indeed,infections represent a significant health risk to various patients,including hospitalised individuals, as well as those with underlyingdisease conditions and/or immune defects. In particular, wounds andinfections represent a serious risk to diabetic patients. These patientsoften experience slow and/or incomplete wound healing, ulceration of theextremities, and are prone to infection. In diabetic patients, ulcersare often large, open wounds that can involve both soft tissue and theunderlying bone. The problem is widespread, as approximately 15% of alldiabetics develop ulcers. Infections within these ulcers are difficultto successfully treat, due to poor circulation at these sites (e.g.,limiting the potential for systemically administered antimicrobialtreatments to reach the wound sites). In extreme cases, limb amputationbecomes necessary. Indeed, these amputations account for half of allamputations done in the U.S. Thus, wounds and ulcers of diabeticpatients often represent life threatening conditions for diabeticsubjects.

Current treatment of diabetic wounds or ulcers consists of debridement,packing the wound with gauze, and placing the patient on systemicantimicrobials. However, no evidence exists that an adequate amount ofdrug reaches the wound site with this treatment. Indeed, there remains aneed for compositions and methods for improved wound healing foradministration to diabetic individuals.

Wound repair is a complex process involving the continual communicationand interaction between fibroblasts, endothelial cells, keratinocytes,inflammatory cells and the extracellular matrix (ECM). Efficient woundrepair requires adequate formation of granulation tissue to maintain asupply of nutrients in the wound area (Arbiser, J. Am. Acad. Dermatol.,34:486–497 [1996]; and Gallit and Clark, Curr. Op. Cell. Biol.,6:717–725 [1994]), and for extracellular matrix deposition (collagensynthesis). When collagen synthesis is impaired or inhibited, woundsheal slowly and incompletely (Streit et al., EMBO 19:3272–3282 [2000];and Goodson and Hunt, J. Surg. Res., 22:221–227 [1997]). Thus, what isneeded are means to facilitate wound healing, particularly inindividuals with impaired and/or incomplete wound healing capabilities,such as diabetic individuals.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions useful inlocalized transfer of genetic material or proteins. Moreover, thepresent invention provides methods and compositions for improving and/orcontrolling wound healing by applying a wound care device comprisingHoxD3 and/or HoxA3 and/or HoxB3. In addition, the present inventionprovides methods and compositions for improved wound healing in subjectshaving impaired healing capabilities, such as diabetic subjects.

Thus, the present invention provides methods of treating a wound,comprising the step of applying a wound care device comprising HoxD3 DNAto a wound. Some embodiments comprise HoxD3 DNA as set forth in SEQ IDNO:1. In preferred embodiments, the applying is done under conditionssuch that wound healing is accelerated, under conditions such that woundclosure is accelerated, under conditions such that angiogenesis in thewound is enhanced or under conditions such that type I collagenexpression in the wound is enhanced. In some embodiments, the wound caredevice further comprises a cellulosic material.

The present invention also provides methods of treating a wound,comprising the step of applying a wound care device comprising HoxD3protein to a wound. Some embodiments comprise HoxD3 protein as set forthin SEQ ID NO:2. In preferred embodiments, the applying is done underconditions such that wound healing is accelerated, under conditions suchthat wound closure is accelerated, under conditions such thatangiogenesis in the wound is enhanced or under conditions such that typeI collagen expression in the wound is enhanced. In some embodiments, thewound care device further comprises a cellulosic material.

In still further embodiments, the present invention provides methods oftreating a wound having impaired healing capabilities, comprising thestep of applying a wound care device comprising HoxD3 DNA to a wound.Some embodiments comprise HoxD3 DNA as set forth in SEQ ID NO:1. Inpreferred embodiments, the wound having impaired healing capabilities isa diabetic wound. In other preferred embodiments, the applying is doneunder conditions such that wound healing is accelerated, underconditions such that wound closure is accelerated, under conditions suchthat angiogenesis in the wound is enhanced, or under conditions suchthat type I collagen expression in the wound is enhanced. In someembodiments, the wound care device further comprises a cellulosicmaterial.

The present invention also provides methods of treating a wound havingimpaired healing capabilities, comprising the step of applying a woundcare device comprising HoxD3 protein to a wound. Some embodimentscomprise HoxD3 protein as set forth in SEQ ID NO:2. In preferredembodiments, the wound having impaired healing capabilities is adiabetic wound. In preferred embodiments, the applying is done underconditions such that wound healing is accelerated, under conditions suchthat wound closure is accelerated, under conditions such thatangiogenesis in the wound is enhanced or under conditions such that typeI collagen expression in the wound is enhanced. In some embodiments, thewound care device further comprises a cellulosic material.

Also provided by the present invention are methods for gene transfer toa localized area, comprising the step of applying a cellulosic materialcomprising a plasmid encoding at least one protein of interest to alocalized area. In some embodiments, cellulosic material comprisesmethylcellulose. In preferred embodiments, the localized area is awound, while in particularly preferred embodiments, the wound is adiabetic wound. In some embodiments, the wound is an ulcer. In preferredembodiments, the protein of interest is a protein involved in woundhealing. In some embodiments, the protein of interest is HoxD3, while inrelated embodiments, the protein of interest is HoxD3 as set forth inSEQ ID NO:2. In other embodiments, the plasmid comprises HoxD3 DNA asset forth in SEQ ID NO:1.

Moreover, the present invention provides methods of treating a wound,comprising the step of applying a wound care device comprising a HoxD10inhibitor to a wound. In preferred embodiments, the applying is doneunder conditions such that wound healing is accelerated, underconditions such that wound closure is accelerated, under conditions suchthat angiogenesis in the wound is enhanced, or under conditions suchthat type I collagen expression in the wound is enhanced. In someembodiments, the wound care device further comprises a cellulosicmaterial. In preferred embodiments, the HoxD10 inhibitor is selectedfrom the group consisting of HoxD10 antisense molecules, HoxD10 dominantnegative mutants, HoxD10 RNAi molecules, HoxD10-reactive antibodies, andHoxD10 artificial substrates.

The present invention also provides methods of treating a wound havingimpaired healing capabilities, comprising the step of applying a woundcare device comprising a HoxD10 inhibitor to a wound. In preferredembodiments, the wound is a diabetic wound. In particularly preferredembodiments, the applying is done under conditions such that woundhealing is accelerated, under conditions such that wound closure isaccelerated, under conditions such that angiogenesis in the wound isenhanced or under conditions such that type I collagen expression in thewound is enhanced. In some embodiments, the wound care device furthercomprises a cellulosic material. In preferred embodiments, the HoxD10inhibitor is selected from the group consisting of HoxD10 antisensemolecules, HoxD10 dominant negative mutants, HoxD10 RNAi molecules,HoxD10-reactive antibodies, and HoxD10 artificial substrates.

Also provided by the present invention are compositions comprising acellulosic material and a gene encoding at least one protein ofinterest. In some embodiments, the protein of interest is HoxD3 as setforth in SEQ ID NO:2. In some embodiments, the HoxD3 is a recombinantHoxD3 protein. In further embodiments, the recombinant HoxD3 protein isa fusion protein.

The present invention also provides compositions comprising a cellulosicmaterial and at least one protein of interest. In some embodiments, theprotein of interest is HoxD3 as set forth in SEQ ID NO:2. In someembodiments, the HoxD3 is a recombinant HoxD3 protein. In furtherembodiments, the recombinant HoxD3 protein is a fusion protein.

In some embodiments, the present invention provides compositionscomprising a cellulosic material and an inhibitor of HoxD10, wherein theinhibitor is an inhibitor of HoxD10 DNA, an inhibitor of HoxD10 RNA, oran inhibitor of HoxD10 protein. In preferred embodiments, the inhibitorof HoxD10 is selected from the group consisting of HoxD10 antisensemolecules, HoxD10 dominant negative mutants, HoxD10 RNAi molecules,HoxD10-reactive antibodies, and HoxD10 artificial substrates.

The present invention further provides compositions comprisingcellulosic material and HoxA3 nucleic acid. In various embodiments, theHoxA3 nucleic acid comprises DNA or RNA, while the cellulosic materialcomprises methylcellulose. In preferred embodiments, the HoxA3 nucleicacid comprises a nucleic acid encoding a protein set forth in SEQ IDNO:15, a biologically active portion thereof or a biologically activevariant thereof. In a subset of these embodiments, the HoxA3 nucleicacid comprises a nucleic acid set forth in SEQ ID NO:14. In particularlypreferred embodiments, the HoxA3 nucleic acid is located in anexpression vector. In various embodiments, the expression vector isselected from the group consisting of a plasmid vector, a recombinantviral vector and a recombinant bacterial vector. Also provided areembodiments in which the composition further comprises HoxD3 nucleicacid. In preferred embodiments, the HoxD3 nucleic acid comprises anucleic acid encoding a protein set forth in SEQ ID NO:2, a biologicallyactive portion thereof or a biologically active variant thereof. Invarious embodiments, the HoxD3 nucleic acid comprises a nucleic acid setforth in SEQ ID NO:1.

Also provided by the present invention are compositions comprisingcellulosic material and HoxA3 protein. In various embodiments, thecellulosic material comprises methylcellulose. In preferred embodiments,the HoxA3 protein comprises a protein set forth in SEQ ID NO:15, abiologically active portion thereof or a biologically active variantthereof. In some embodiments, the HoxA3 protein is a recombinant HoxA3protein. In a subset of these embodiments, the recombinant HoxA3 proteinis a fusion protein comprising an affinity tag. Also provided areembodiments in which the composition further comprises HoxD3 protein. Inpreferred embodiments, the HoxD3 protein comprises a protein set forthin SEQ ID NO:2, a biologically active portion thereof or a biologicallyactive variant thereof.

Moreover, the present invention provides methods comprising: providing;a subject with a wound, and a composition comprising HoxA3 nucleic acidor HoxA3 protein; and applying said composition to said wound. Invarious embodiments, the HoxA3 nucleic acid comprises DNA or RNA. Insome preferred embodiments, the HoxA3 nucleic acid comprises a nucleicacid encoding a protein set forth in SEQ ID NO:15, a biologically activeportion thereof or a biologically active variant thereof. In otherpreferred embodiments, the HoxA3 protein comprises a protein set forthin SEQ ID NO:15 or a biologically active portion thereof, a biologicallyactive portion thereof or a biologically active variant thereof. Alsoprovided are embodiments of the present invention in which the applyingis under conditions such that wound healing is accelerated, the applyingis under conditions such that wound closure is accelerated, the applyingis under conditions such that angiogenesis in said wound is enhanced,and/or the applying is under conditions such that type I collagenexpression in said wound is enhanced. In some embodiments, thecomposition further comprises a cellulosic material. In variousembodiments, the cellulosic material comprises methylcellulose. Inpreferred embodiments, the composition is located in a wound caredevice. Also provided are embodiments in which the wound has impairedhealing capabilities. In preferred embodiments, the wound havingimpaired healing capabilities is a diabetic wound. In some embodiments,the wound is an ulcer. Also provided are embodiments in which thecomposition further comprises HoxD3 nucleic acid or HoxD3 protein. Invarious embodiments, the HoxD3 nucleic acid comprises a nucleic acidencoding a protein set forth in SEQ ID NO:2, a biologically activeportion thereof or a biologically active variant thereof. In otherembodiments, the HoxD3 protein comprises a protein set forth in SEQ IDNO:2, a biologically active portion thereof or a biologically activevariant thereof. In a subset of these embodiments, the HoxD3 nucleicacid comprises a nucleic acid set forth in SEQ ID NO:1.

The present invention provides compositions comprising cellulosicmaterial and HoxB3 nucleic acid. In some embodiments, the HoxB3 nucleicacid comprises a nucleic acid encoding a protein set forth in SEQ IDNO:23, a biologically active portion thereof, or a biologically activevariant thereof. In a subset of these embodiments, the HoxB3 nucleicacid comprises a nucleic acid set forth in SEQ ID NO:22. In somepreferred embodiments, the HoxB3 nucleic acid is located in anexpression vector. In additional embodiments, the composition furthercomprises one or both of HoxD3 nucleic acid and HoxA3 nucleic acid. Insome embodiments, the HoxD3 nucleic acid comprises a nucleic acidencoding a protein set forth in SEQ ID NO:2, a biologically activeportion thereof, or a biologically active variant thereof, and the HoxA3nucleic acid comprises a nucleic acid encoding a protein set forth inSEQ ID NO:15, a biologically active portion thereof, or a biologicallyactive variant thereof.

Furthermore, the present invention provides compositions comprisingcellulosic material and HoxB3 protein. In some embodiments, the HoxB3protein comprises a protein set forth in SEQ ID NO:23, a biologicallyactive portion thereof, or a biologically active variant thereof. Insome embodiments, the HoxB3 protein is a recombinant HoxB3 protein. In asubset of these embodiments, the recombinant HoxB3 protein is a fusionprotein comprising an affinity tag. In additional embodiments, thecomposition further comprises one or both of HoxD3 protein and HoxA3protein. In some preferred embodiments, the HoxD3 protein comprises aprotein set forth in SEQ ID NO:2, a biologically active portion thereof,or a biologically active variant thereof, and the HoxA3 proteincomprises a protein set forth in SEQ ID NO:15, a biologically activeportion thereof, or a biologically active variant thereof.

The present invention also provides methods comprising: providing asubject with a wound, and a composition comprising HoxB3 nucleic acid orHoxB3 protein; and applying the composition to the wound. In someembodiments, the HoxB3 nucleic acid comprises a nucleic acid encoding aprotein set forth in SEQ ID NO:23, a biologically active portion thereofor a biologically active variant thereof, while the HoxB3 proteincomprises a protein set forth in SEQ ID NO:23, a biologically activeportion thereof or a biologically active variant thereof. In somepreferred embodiments, the applying is under conditions such that woundclosure is accelerated, and/or the applying is under conditions suchthat angiogenesis in the wound is enhanced. In some preferredembodiments, the composition further comprises a cellulosic material. Inpreferred embodiments, the wound has impaired healing capabilities, andin a subset of these, the wound having impaired healing capabilities isa diabetic wound. Moreover, in some embodiments the composition furthercomprises one or both of HoxD3 nucleic acid and HoxA3 nucleic acid, orone or both of HoxD3 protein and HoxA3 protein.

DESCRIPTION OF THE FIGURES

FIG. 1 provides photographs of trichrome-stained tissue sections showingcollagen deposition in db/db wounds 14 days after creation of 1 cm openwounds. Panel A provides a low power image of a db/db wound treated withcontrol DNA. As indicated in this Panel, there is limited collagendeposition (shown in blue). Panel B provides a higher power image ofPanel A. Panel C provides a low power image of a HoxD3 DNA treatedwound. As indicated in this Panel, there is more extensive collagendeposition in the HoxD3 DNA treated wound, as compared to the control.Panel D provides a higher power image of Panel C, showing extensivecollagen deposition and the presence of small microvessels in thetreated wound.

FIG. 2 provides a graph showing closure of 2.5 cm wounds in diabetic(db/db) mice treated with either control DNA or HoxD3 DNA. The observeddifferences in wound closure are statistically significant at all timepoints between 7 and 49 days post-wounding.

FIG. 3 provides photographs of two animals at 7 and 14 days afterwounding (0.8 cm wounds). As indicated by these photographs, the woundclosure was significantly improved with the HoxD3 DNA treatment, ascompared to the control DNA treatment.

FIG. 4 provides Northern blot analyses of type I collagen RNA expressionin control and HoxD3 DNA-treated 0.8 cm wounds. Panel A shows expressionof type I collagen (Col1A1) mRNA and corresponding total RNA loading(rRNA) in bilateral wounds from a diabetic (db/db) mouse treated withcontrol or HoxD3 DNA for 7 days. Panel B shows that expression of type Icollagen mRNA remains higher in tissues taken from HoxD3 DNA-treatedwounds, as compared to bilateral control-treated wounds from the sameanimals as described for Panel A, after 10 days. The correspondingribosomal RNA (rRNA) loading controls are shown below. Panel C showsthat expression of type I collagen mRNA remains higher in HoxD3DNA-treated wounds as compared to corresponding control DNA-treatedwounds from the same animals as described for Panels A and B, after 17days.

FIG. 5 provides photographs of trichrome-stained tissue sections showingcollagen deposition (in blue) in db/db wounds 21 and 42 days aftercreation of 0.8 cm open wounds. Panel A depicts a healing wound 21 daysfollowing application of HoxD3 DNA. Panel B provides a highermagnification of the 21 day wound demonstrating the appearance oforganized collagen fibrils and numerous small capillaries. Panel Cdepicts a healing wound 42 days after HoxD3 DNA transfer. Panel Dprovides a higher magnification of the 42 day wound demonstrating theappearance of collagen fibrils and small blood vessels.

FIG. 6 provides photographs of chick CAM grafted with fibrosarcoma cellstransfected with CK (control) or HoxA3. Panel A is a low power image ofa chick CAM whole mount taken 72 hours after grafting withCK-transfected fibrosarcoma cells, while Panel B is a higher powerphotomicrograph which fails to reveal any significant branching orangiogenesis in the tissue adjacent to the tumor. Panel C is a low powerphotomicrograph of CAM tissue 72 hours after receiving fibrosarcomacells shedding HoxA3 retrovirus, while Panel D is a higher powerphotomicrograph showing extensive branching of small microvesselsadjacent to the tumor mass.

FIG. 7 provides photomicrographs of fibrosarcoma grafts infected with arecombinant retrovirus expressing human HoxA3. Panel A is an in situhybridization image revealing the expression of retrovirally producedHoxA3 mRNA at the edge of the tumor (arrows) and extending into theadjacent CAM tissue. (Bar=100 μM). Panel B is a higher powerphotomicrograph showing HoxA3 mRNA in EC (arrows) of a small vessel (v)adjacent to the tumor. (Bar=20 μM). Panel C shows DAPI nuclear stainingof a serial section taken from CAM tissue, which had been grafted withtumor cells shedding HoxA3 retrovirus. Panel D shows immunofluorescensestaining of the same serial section as shown in panel C, with apolyclonal antibody against the endothelial cell marker, von Willebrandfactor.

FIG. 8 depicts the influence of HoxA3 on gene expression in endothelialcells. Panel A (top) shows northern blot analysis of urokinaseplasminogen activator receptor (uPAR) mRNA levels in HMEC-1 transfectedwith control plasmid or with HoxA3 or HoxD3 expression vectors. The blotwas stripped and reprobed for expression of MMP-14 (middle), while therelative loading of each sample as visualized by ethidium bromidestaining of total ribosomal RNA (bottom). Panel B shows a western blotanalysis of uPAR (upper) and MMP-14 (lower) protein expression in totalcell lysate harvested from control or HoxA3 transfected cells. Panel C(top) shows a northern blot analysis of MMP-2 expression in control,HoxA3, and HoxD3 transfected HMEC-1, and relative RNA loading (bottom).Panel D is a gelatin zymograph of conditioned media collected fromcontrol transfected cells (HMEC), co-cultures of HoxA3 and HoxD3transfected cells (HoxA3+D3), HMEC and HoxA3 transfected cells (HoxA3)or HMEC and HoxD3 (HoxD3) co-cultures. The stronger upper bandcorresponds to the 68 kD inactive pro-MMP-2 and the lower band showsproteolysis arising from the approximately 62 kD active form of MMP-2.

FIG. 9 indicates that HoxA3 promotes EC migration in fibrin. Thephotomicrographs in panels A and B show the degree of migration observedfor each condition, 72 hours following embedding into three-dimensionalfibrin gels (Bar=10 μM). Panel C shows the extent of migration ofcontrol, HoxA3 and HoxD3-transfected HMEC-1 cells after 5 hours inmodified Boyden chambers coated with 20 mg/ml fibrinogen. Data areexpressed as the mean+/−standard deviation (n=3), and ** denotesstatistical significance p<0.05.

FIG. 10 depicts the relationship between basement membrane contact(panel A), bFGF treatment (panel B), and wounding (panels C and D) onHoxA3 expression (Bar=10 μM).

FIG. 11 demonstrates that HoxA3 expression promotes wound repair indiabetic mice. Panels A and B show the appearance of 2.5 cm wounds, 14days following application of control cDNA or HoxA3 expression plasmids,respectively. Panel C provides a graph demonstrating that HoxA3treatment enhances wound closure rate in diabetic mice. Squaresrepresent the diameter of control-treated wounds over time, whereas thediamonds represent the diameter of Hox A3-treated wounds over time. Micetreated with HoxA3 showed a significantly greater (**p<0.05) degree ofclosure at days 7 through 42, as compared to control DNA treated wounds(n=10).

FIG. 12, panels A–C, provide photomicrographs of H and E stained healedtissue sections 21 days after HoxA3 application.

FIG. 13 provides images of control (panel A), HoxA3+ (panel B), andHoxD3+ (panel C) keratinocytes 48 hours after application of scratchwounds. Panel D is a quantitative analysis of relative numbers of cellsmigrating into the wound area 72 hours after scratch wounding.

FIG. 14 provides a graph demonstrating that HoxB3 treatment enhances thewound closure rate in diabetic mice. Squares represent the diameter ofcontrol-treated (βgal DNA) wounds over time, whereas the diamondsrepresent the diameter of HoxB3-treated (HoxB3 expression vector) woundsover time. Also shown are the wound closure times of wild type mice, aswell as diabetic mice treated with HoxD3, and HoxA3.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of a polypeptideor precursor or RNA (e.g., tRNA, siRNA, rRNA, etc.). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction, etc.) ofthe full-length or fragment are retained. The term also encompasses thecoding region of a structural gene and the sequences located adjacent tothe coding region on both the 5′ and 3′ ends, such that the genecorresponds to the length of the full-length mRNA. The sequences thatare located 5′ of the coding region and which are present on the mRNAare referred to as 5′ untranslated sequences. The sequences that arelocated 3′ or downstream of the coding region and that are present onthe mRNA are referred to as 3′ untranslated sequences. The term “gene”encompasses both cDNA and genomic forms of a gene. A genomic form orclone of a gene contains the coding region, which may be interruptedwith non-coding sequences termed “introns” or “intervening regions” or“intervening sequences.” Introns are removed or “spliced out” from thenuclear or primary transcript, and are therefore absent in the messengerRNA (mRNA) transcript. The mRNA functions during translation to specifythe sequence or order of amino acids in a nascent polypeptide.

In particular, the term “HoxD3 gene” refers to the full-length HoxD3nucleotide sequence. However, it is also intended that the termencompass fragments of the HoxD3 nucleotide sequence, as well as otherdomains (e.g., functional domains) within the full-length HoxD3nucleotide sequence. Furthermore, the terms “HoxD3 gene,” “HoxD3nucleotide sequence,” and “HoxD3 polynucleotide sequence” encompass DNA,cDNA, and RNA sequences.

The term “plasmid” as used herein, refers to a small, independentlyreplicating, piece of DNA. Similarly, the term “naked plasmid” refers toplasmid DNA devoid of extraneous material typically used to affecttransfection. As used herein, a “naked plasmid” refers to a plasmidsubstantially free of calcium-phosphate, DEAE-dextran, liposomes, and/orpolyamines.

As used herein, the term “purified” refers to molecules (polynucleotidesor polypeptides) that are removed from their natural environment,isolated or separated. “Substantially purified” molecules are at least50% free, preferably at least 75% free, and more preferably at least 90%free from other components with which they are naturally associated.

The term “recombinant DNA” refers to a DNA molecule that is comprised ofsegments of DNA joined together by means of molecular biologytechniques. Similarly, the term “recombinant protein” refers to aprotein molecule that is expressed from recombinant DNA.

The term “fusion protein” as used herein refers to a protein formed byexpression of a hybrid gene made by combining two gene sequences.Typically this is accomplished by cloning a cDNA into an expressionvector in frame with an existing gene. The fusion partner may act as areporter (e.g., βgal) or may provide a tool for isolation purposes(e.g., GST).

Suitable systems for production of recombinant proteins include but arenot limited to prokaryotic (e.g., Escherichia coli), yeast (e.g.,Saccaromyces cerevisiae), insect (e.g., baculovirus), mammalian (e.g.,Chinese hamster ovary), plant (e.g., safflower), and cell-free systems(e.g., rabbit reticulocyte).

As used herein, the term “coding region” refers to the nucleotidesequences that encode the amino acid sequences found in the nascentpolypeptide as a result of translation of an mRNA molecule. The codingregion is bounded in eukaryotes, on the 5′ side by the nucleotidetriplet “ATG” that encodes the initiator methionine and on the 3′ sideby one of the three triplets which specify stop codons (i.e., TAA, TAG,and TGA).

Where amino acid sequence is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, “amino acidsequence” and like terms, such as “polypeptide” or “protein,” are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule. The term“wild-type” refers to a gene or gene product that has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene.

In contrast, the terms “modified,” “mutant,” and “variant” refer to agene or gene product that displays modifications in sequence and orfunctional properties (i.e., altered characteristics) when compared tothe wild-type gene or gene product. It is noted that naturally occurringmutants can be isolated; these are identified by the fact that they havealtered characteristics when compared to the wild-type gene or geneproduct.

As used herein, the term “homeobox” refers to a conserved DNA sequenceoriginally detected by DNA hybridization in many of the genes that giverise to homeotic and segmentation mutants in Drosophila. In particular,the homeobox consists of about 180 nucleotides coding for a sequence ofabout 60 amino acids, sometimes termed the homeodomain, which isinvolved in binding to DNA.

The terms “HoxD3,” “Hox D3,” “homeobox D3,” “Hox4A,” and “Hox-4.1,” asused herein refer to a human homeobox gene (e.g., Homo sapiens—GenBankAccession No. D1117) and its gene product, as well as its vertebratecounterparts, including wild type and mutant products. The human HoxD3coding region is set forth as SEQ ID NO:1, while the human HoxD3 proteinsequence is set forth as SEQ ID NO:2. Moreover, the human HoxD3 cDNAsequence is set forth as SEQ ID NO:12, and the human HoxD3 gene is setforth as SEQ ID NO:13. Vertebrate counterparts of HoxD3 includemammalian HoxD3 (e.g., Mus musculus—GenBank Accession No. NM_(—)010468),avian HoxD3 (e.g., Gallus gallus—GenBank Accession No. AF067959),reptilian HoxD3, amphibian HoxD3, piscean HoxD3, marsipobranchian HoxD3,and leptocardian HoxD3. Preferred embodiments of the present inventioncomprise mammalian HoxD3. HoxD3 variants, which differ from the wildtype HoxD3 sequences in fewer than 1% of the residues, may also besuitable for use in the methods and compositions of the presentinvention.

As used herein, the terms “HoxA3,” “Hox A3,” “homeobox A3,” “Hox1E” and“Hox-1.5-like” refer to a human homeobox gene (e.g., Homosapiens—GenBank Accession No. AC004079) and its gene product, as well asits vertebrate counterparts, including wild type and mutant products.The human HoxA3 coding region is set forth as SEQ ID NO:14, while thehuman HoxA3 protein sequence is set forth as SEQ ID NO:15. Moreover, thehuman HoxA3 cDNA sequence is set forth in SEQ ID NO:16. Vertebratecounterparts of HoxA3 include mammalian HoxA3 (e.g., Musmusculus—GenBank Accession Nos. XM_(—)13275), avian HoxA3, reptilianHoxA3, amphibian HoxA3, piscean HoxA3 (e.g., Danio rerio—GenBankAccession No. NM_(—)131534; and Heterodontus francisci—GenBank AccessionNo. AF224262), marsipobranchian HoxA3, and leptocardian HoxA3. Preferredembodiments of the present invention comprise the “a isoform” of amammalian HoxA3. HoxA3 variants, which differ from the wild type HoxA3sequences in fewer than 1% of the residues, may also be suitable for usein the methods and compositions of the present invention. In particular,HoxA3 variants including but not limited to those of GenBank AccessionNos. NM_(—)030661, NM_(—)153631, and NM_(—)153632, as well asnon-naturally occurring variants generated by recombinant or other means(e.g. amino acid substitution, deletion, or addition) are contemplatedto find use in the compositions and methods of the present invention.

As used herein, the terms “HoxB3,” “Hox B3,” “homeobox B3,”“Hox2,”“Hox2G,” and “Hox-2.7,” refer to a human homeobox gene (e.g.,Homo sapiens—GenBank Accession No. NM_(—)002146) and its gene product,as well as its vertebrate counterparts, including wild type and mutantproducts. The human HoxB3 coding region is set forth as SEQ ID NO:22,while the human HoxB3 protein sequence is set forth as SEQ ID NO:23.Moreover, the human HoxB3 cDNA sequence is set forth in SEQ ID NO:24.Vertebrate counterparts of HoxB3 include mammalian HoxB3 (e.g., Musmusculus—GenBank Accession No. NM_(—)010458), avian HoxB3 (e.g., Gallusgallus—GenBank Accession No. X74506), reptilian HoxB3, amphibian HoxB3(e.g., Pleurodeles waltl—AY383550), piscean HoxB3 (e.g., Daniorerio—GenBank Accession No. AJ537509), marsipobranchian HoxB3, andleptocardian HoxB3. HoxB3 variants that differ from the wild type HoxB3sequences in fewer than 1% of the residues, may also be suitable for usein the methods and compositions of the present invention. In particular,naturally occurring HoxB3 variants, including but not limited to thoseof GenBank Accession No. NM_(—)002146, as well as non-naturallyoccurring variants, generated by recombinant or other means (e.g. aminoacid substitution, deletion, or addition) are contemplated to find usein the compositions and methods of the present invention.

The terms “HoxD10” and “Hox4D” refer to another human homeobox gene(GenBank Accession No. NM_(—)002148) and its gene product, as well asits vertebrate counterparts. The human HoxD10 coding region is set forthas SEQ ID NO:9, while the human HoxD10 protein sequence is set forth asSEQ ID NO:10, and the human HoxD10 cDNA sequence is set forth as SEQ IDNO:11. Preferred embodiments of the present invention comprise mammalianHoxD10. HoxD10 variants, which differ from the wild type HoxD10sequences in fewer than 1% of the residues, may also be suitable for usein the methods and compositions of the present invention.

As used herein, the term “HoxD10 inhibitor” refers to any molecule,which reduces the expression of or activity of HoxD10. HoxD10 inhibitorssuitable for use in the methods and compositions of the presentinvention include but are not limited to antisense molecules, RNAimolecules, HoxD10-reactive antibodies, dominant negative mutants, andartificial substrates.

The term “antisense molecule” refers to polynucleotides andoligonucleotides capable of binding to an mRNA molecule. In particular,an antisense molecule is a DNA or RNA sequence complementary to an mRNAsequence of interest. In preferred embodiments, the term HoxD10antisense molecule refers to a single-stranded DNA or RNA sequence thatbinds to at least a portion of a HoxD10 mRNA molecule to form a duplexwhich then blocks further transcription and/or translation.

As used herein, the terms “complementary” and “complementarity” refer topolynucleotides related by base-pairing rules. For example, for thesequence “5′-AGT-3′,” the complementary sequence is “3′-TCA-5′.”

The term “RNAi” refers to a double stranded RNA molecule, with eachstand consisting of at least 20 nucleotides, which direct thesequence-specific degradation of mRNA through a process known as RNAinterference. Thus RNAi can be used to block gene expressionposttranscriptionally (Zamore et al., Cell 101: 25–33 [2000]).

The term “antibody” refers to polyclonal and monoclonal antibodies.Polyclonal antibodies which are formed in the animal as the result of animmunological reaction against a protein of interest or a fragmentthereof, can then be readily isolated from the blood using well-knownmethods and purified by column chromatography, for example. Monoclonalantibodies can also be prepared using known methods (See, e.g., Winterand Milstein, Nature, 349, 293–299 [1991]). As used herein, the term“antibody” encompasses recombinantly prepared, and modified antibodiesand antigen-binding fragments thereof, such as chimeric antibodies,humanized antibodies, multifunctional antibodies, bispecific oroligo-specific antibodies, single-stranded antibodies and F(ab) orF(ab)₂ fragments (See, e.g., EP-B1-0 368 684, U.S. Pat. No. 4,816,567,U.S. Pat. No. 4,816,397, WO 88/01649, WO 93/06213, WO 98/24884). Theterm “reactive” in used in reference to an antibody indicates that theantibody is capable of binding an antigen of interest. For example, aHoxD10-reactive antibody is an antibody, which binds to Hox10 or to afragment of HoxD10.

The term “dominant negative mutant” refers to molecules that lack wildtype activity, but which effectively compete with wild type moleculesfor substrates, receptors, etc., and thereby inhibit the activity of thewild type molecule. In preferred embodiments, the term “HoxD10 dominantnegative mutant” refers to a HoxD10 mutant protein which competes withthe wild type HoxD10 protein for DNA substrates, but which fails toinduce downstream effects.

As used herein, the term “artificial substrate” refers to syntheticsubstance upon which a molecule of interest acts. In some embodiments,the term “HoxD10 artificial substrate” refers to molecules that bindHoxD10 to the exclusion of native HoxD10 substrates. Preferred “HoxD10substrates” include but are not limited to DNA fragments to which HoxD10binds. The term “DNA fragment” refers to pieces of DNA that are not partof the genome.

The term “portion” when used in reference to a nucleotide sequencerefers to fragments of that sequence, which range in size from 10nucleotides to the entire nucleotide sequence minus one nucleotide.

As used herein, the term “biologically active” refers to a moleculehaving structural, regulatory and or biochemical functions of a wildtype homeobox molecule. In some instances, the biologically activemolecule is a homolog of a mammalian homeobox molecule, while in otherinstance the biologically active molecule is a portion of a mammalianhomeobox molecule. Other biologically active molecules, which find usein the compositions and methods of the present invention include but arenot limited to mutant (e.g., variants with at least one deletion,insertion or substitution) mammalian homeobox molecules. Biologicalactivity is determined for example, by restoration or introduction ofHox (e.g., HoxA3 or HoxD3) activity in cells which lack Hox activity,through transfection of the cells with a Hox expression vectorcontaining a Hox gene, derivative thereof, or portion thereof. Methodsuseful for assessing HoxA3 activity include but are not limited toRT-PCR for induction of uPAR or MMP14 expression in transfected EC,assessment of angiogenesis of transfected EC grafted onto CAM, andmigration of transfected EC in fibrin gels. Similarly, methods usefulfor assessing HoxD3 activity include but are not limited to Northernanalysis or RT-PCR for induction of type I collagen or MMP-2 expressionin transfected EC cells, and migration of transfected EC on fibrinogensurfaces.

The term “conservative substitution” as used herein refers to a changethat takes place within a family of amino acids that are related intheir side chains. Genetically encoded amino acids can be divided intofour families: (1) acidic (aspartate, glutamate); (2) basic (lysine,arginine, histidine); (3) nonpolar (alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan); and (4)uncharged polar (glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine aresometimes classified jointly as aromatic amino acids. In similarfashion, the amino acid repertoire can be grouped as (1) acidic(aspartate, glutamate); (2) basic (lysine, arginine, histidine), (3)aliphatic (glycine, alanine, valine, leucine, isoleucine, serine,threonine), with serine and threonine optionally be grouped separatelyas aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine,tryptophan); (5) amide (asparagine, glutamine); and (6)sulfur-containing (cysteine and methionine) (e.g., Stryer ed.,Biochemistry, pg. 17–21, 2nd ed, WH Freeman and Co. [1981]). Whether achange in the amino acid sequence of a peptide results in a functionalhomolog can be readily determined by assessing the ability of thevariant peptide to function in a fashion similar to the wild-typeprotein. Peptides having more than one replacement can readily be testedin the same manner. In contrast, the term “nonconservative substitution”refers to a change in which an amino acid from one family is replacedwith an amino acid from another family (e.g., replacement of a glycinewith a tryptophan). Guidance in determining which amino acid residuescan be substituted, inserted, or deleted without abolishing biologicalactivity can be found using computer programs (e.g., LASERGENE software,DNASTAR Inc., Madison, Wis.).

The terms “mammals” and “mammalian” refer animals of the class mammalia,which nourish their young by fluid secreted from mammary glands of themother, including human beings. The class “mammalian” includes placentalanimals, marsupial animals, and monotrematal animals. Preferredembodiments of the present invention include but are not limited to amammalian HoxD3 gene or gene product (e.g., mice, rats, pigs, monkeys,humans, etc.). In a particularly preferred embodiment of the presentinvention, the term “HoxD3 DNA” refers to the open reading frame orcoding region of HoxD3 gene from Homo sapiens (SEQ ID NO:1), while theterm “HoxD3 protein” refers to the amino acid sequence encoded by theHoxD3 DNA of Homo sapiens (SEQ ID NO:2).

As used herein the term “animal” refers to any member of the kingdomAnimalia, which includes living things, which have cells differing fromplant cells with regard to the absence of a cell wall and chlorophylland the capacity for spontaneous movement. Preferred embodiments of thepresent invention are primarily directed to vertebrate (backbone ornotochord) members of the animal kingdom.

The term “diabetic” as used here refers to organisms which have adisorder characterized by the insufficient production or utilization ofinsulin. Insulin is a pancreatic hormone that is needed to convertglucose for cellular metabolism and energy production. In preferredembodiments of the present invention, the term “diabetic patient” refersto patients suffering from diabetes mellitus. The term “diabetic”encompasses both patients with type I diabetes Ouvenile onset) andpatients with type II diabetes (adult onset). “Type I diabetes” alsoreferred to as “insulin-dependent diabetes” is a form of diabetesmellitus that usually develops during childhood or adolescence and ischaracterized by a severe deficiency in insulin secretion resulting fromatrophy of the islets of Langerhans and causing hyperglycemia and amarked tendency towards ketoacidosis. “Type II diabetes” also referredto as “non-insulin-dependent diabetes” is a form of diabetes mellitusthat develops especially in adults (most often in obese individuals) andthat is characterized by hyperglycemia resulting from bothinsulin-resistance and an inability to produce more insulin.

The terms “patient” and “subject” refer to a mammal or an animal who isa candidate for receiving medical treatment.

As used herein, the term “wound” refers to a disruption of the normalcontinuity of structures caused by a physical (e.g., mechanical) force,a biological (e.g., thermic or actinic force, or a chemical means. Inparticular, the term “wound” encompasses wounds of the skin. The term“wound” also encompasses contused wounds, as well as incised, stab,lacerated, open, penetrating, puncture, abrasions, grazes, bums,frostbites, corrosions, wounds caused by ripping, scratching, pressure,and biting, and other types of wounds. In particular, the termencompasses ulcerations (i.e., ulcers), preferably ulcers of the skin.

As used herein, the term “wound healing” refers to a regenerativeprocess with the induction of an exact temporal and spatial healingprogram comprising wound closure and the processes involved in woundclosure. The term “wound healing” encompasses but is not limited to theprocesses of granulation, neovascularization, fibroblast, endothelialand epithelial cell migration, extracellular matrix deposition,re-epithelialization, and remodeling.

The term “wound closure” refers to the healing of a wound wherein sidesof the wound are rejoined to form a continuous barrier (e.g., intactskin).

The term “granulation” refers to the process whereby small, red,grainlike prominences form on a raw surface (that of wounds or ulcers)as healing agents.

The term “neovascularization” refers to the new growth of blood vesselswith the result that the oxygen and nutrient supply is improved.Similarly, the term “angiogenesis” refers to the vascularization processinvolving the development of new capillary blood vessels.

The term “cell migration” refers to the movement of cells (e.g.,fibroblast, endothelial, epithelial, etc.) to the wound site.

The term “extracellular matrix deposition” refers to the secretion bycells of fibrous elements (e.g., collagen, elastin, reticulin), linkproteins (e.g., fibronectin, laminin), and space filling molecules(e.g., glycosaminoglycans). As used herein, the term “type I collagen”refers to the most abundant collagen, which forms large well-organizedfibrils having high tensile strength.

The term “re-epithelialization” refers to the reformation of epitheliumover a denuded surface (e.g., wound).

The term “remodeling” refers to the replacement of and/ordevascularization of granulation tissue.

The term “impaired healing capabilities” comprises wounds, which arecharacterized by a disturbed wound healing process. Examples of woundswith impaired healing capabilities are wounds of diabetic patients andalcoholics, wounds which are infected by microorganisms, ischemicwounds, wounds of patients suffering from deficient blood supply orvenous stasis, and ulcers. Particularly preferred wounds are diabeticwounds.

The term “diabetic wounds” refers to wounds of mammals and humanssuffering from diabetes. An example of a diabetic wound is an ulcer(e.g., Ulcus cruris arteriosum or Necrobiosis lipoidica).

As used herein, the term “ulcer” (i.e., “ulceration”) refers to a localdefect or excavation of the surface of an organ or tissue, produced bysloughing of necrotic tissue. The term encompasses various forms ofulcers (e.g., diabetic, neuropathic, arterial, decubitus, dental,perforating, phagedenic, rodent, trophic, tropical, varicose, venereal,etc.), although in preferred embodiments, surface (i.e., skin) ulcersare involved in the present invention. Especially preferred ulcers arediabetic ulcers.

The term “protein involved in wound healing” refers to any proteindirectly or indirectly involved in curing an injury to the body. Forexample, proteins involved in wound healing include but are not limitedto HoxD3 protein, collagen, PDGF, VEGF, bFGF, and TGFβ.

In some embodiments, the present invention provides methods andcompositions for “accelerating wound healing,” whereby different aspectsof the wound healing process are “enhanced.” As used herein, the term“enhanced” indicates that the methods and compositions provide anincreased wound healing rate. In preferred embodiments, the term“enhanced” indicates that the wound healing rate and/or a wound healingprocess occurs at least 10% faster than is observed in untreated orcontrol-treated wounds. In particularly preferred embodiments, the term“enhanced” indicates that the wound healing rate and/or a wound healingprocess occurs at least 15% faster than is observed in untreated orcontrol-treated wounds. In still further preferred embodiments, the term“enhanced” indicates that the wound healing rate and/or a wound healingprocess occurs at least 20% (e.g., 50%, 100%, . . . ) faster than woundsuntreated or control-treated wounds.

The term “control” refers to subjects or samples which provide a basisfor comparison for experimental subjects or samples. For instance, theuse of control subjects or samples permits determinations to be maderegarding the efficacy of experimental procedures. In some embodiments,the term “control subject” refers to animals, which receive a mocktreatment (e.g., βgal plasmid DNA).

As used herein, the terms “gene transfer” and “transfer of geneticinformation” refer to the process of moving a gene or genes from oneplace to another. In preferred embodiments of the present invention, theterm “gene transfer” refers to the transfer of a polynucleotide to cellsand/or tissues of an animal to achieve a therapeutic effect. In someembodiments, the polynucleotide may be in the form of a plasmid, a genefragment or an oligonucleotide. In some embodiments, “gene transfer” istemporary or transient, in other embodiments “gene transfer” issustained, and in still further embodiments, the gene transfer islong-lived, permanent or stable.

As used herein, “gene transfer” may affect the transfection of cellsand/or tissues. The term “transfection” refers to the introduction offoreign DNA into eukaryotic cells.

As used herein, the terms “localized” and “local” refer to theinvolvement of a limited area. Thus, in contrast to “systemic”treatment, in which the entire body is involved, usually through thevascular and/or lymph systems, localized treatment involves thetreatment of a specific, limited area. Thus, in some embodiments,discrete wounds are treated locally using the methods and compositionsof the present invention.

As used herein, the term “topically” means application to the surface ofthe skin, mucosa, viscera, etc. Similarly, the terms “topically activedrug” and “topically active agent” refer to a substance or composition,which elicits a pharmacologic response at the site of application (e.g.,skin), but is not necessarily an antimicrobial agent.

As used herein, the terms “systemically active drug” and “systemicallyactive agent” are used broadly to indicate a substance or compositionthat will produce a pharmacologic response at a site remote from thepoint of application.

As used herein, the term “cellulosic material” refers to any compositionthat comprises cellulose or cellulose-like material. “Cellulose”(C₆H₁₀O₅)_(x) refers to a polymer of glucose with over 3500 repeat unitsin a chain with β glycoside linkages. In preferred embodiments, thecellulose-like material of the present invention is methylcellulose. Ina particularly preferred embodiment, the cellulose-like material iscarboxymethylcellulose. However, any cellulosic material finds use withthe present invention, as long as the material is suitable forimmobilizing plasmid DNA at a desired site. In addition, the preferredcellulosic material of the present invention is sufficiently small insize to fit in wound sites as appropriate and has hydration-limitingproperties.

As used herein, the term “medical devices” includes any material ordevice that is used on, in, or through a patient's body in the course ofmedical treatment for a disease or injury. Medical devices include, butare not limited to, such items as medical implants, wound care devices,drug delivery devices, and body cavity and personal protection devices.The medical implants include, but are not limited to, urinary catheters,intravascular catheters, dialysis shunts, wound drain tubes, skinsutures, vascular grafts, implantable meshes, intraocular devices, heartvalves, and the like.

As used herein, “wound care devices” include, but are not limited toconventional materials such as dressings, plasters, compresses or gelscontaining the pharmaceuticals that can be used in accordance with thepresent invention. Thus, it is possible to administer the wound caredevices comprising HoxD3 (and/or HoxA3) genes or proteins topically andlocally in order to exert an immediate and direct effect on woundhealing. The topical administration of wound care devices can beeffected, for example, in the form of a solution, an emulsion, a cream,an ointment, a foam, an aerosol spray, a gel matrix, a sponge, drops orwashings. Suitable additives or auxiliary substances are isotonicsolutions, such as physiological sodium chloride solutions or sodiumalginat, demineralized water, stabilizers, collagen containingsubstances such as Zyderm II or matrix-forming substances such aspovidone. To generate a gel basis, formulations, such as aluminumhydroxide, polyacrylacid derivatives (e.g., Carbopol®), and cellulosederivatives (e.g., carboxymethyl cellulose) are suitable. These gels canbe prepared as hydrogels on a water basis or as oleogels with low andhigh molecular weight paraffines or vaseline and/or yellow or white wax.As emulsifier alkali soaps, metal soaps, amine soaps or partial fattyacid esters of sorbitants can be used, whereas lipids can be added asvaseline, natural and synthetic waxes, fatty acids, mono-, di-,triglycerides, paraffin, natural oils (e.g., cocos oil), or syntheticfats (e.g., Miglyol®). The wound care devices comprising HoxD3 genes orproteins according to the invention can also, where appropriate, beadministered topically and locally, in the region of the wound, in theform of liposome complexes or gold particle complexes. This form ofadministration is preferred for vectors which are applicable in genetherapy and which contain a nucleic acid, which can be used inaccordance with the invention.

Furthermore, the treatment can be effected using a transdermaltherapeutic system (TTS), which enables the pharmaceuticals of thepresent invention to be released in a temporally controlled manner. Toimprove the penetration of the administered drug through the membrane,additives such as ethanol, urea or propylene glycol can be added inaddition to polymeric auxiliaries, such as Eudragit®. TTS have beendisclosed, for example, in EP 0 944 398 A1, EP 0 916 336 A1, EP 0 889723 A1 or EP 0 852 493 A1 (all of which are herein incorporated byreference).

The wound care devices comprising HoxD3 (and/or HoxA3) genes or proteinsaccording to the invention can also encompass a cell (e.g., akeratinocyte) expressing a polypeptide of the invention, which is thensecreted into the wound site. A suitable carrier for administering thosemodified cells would be a micro carrier consisting of biocompatiblematerials, such as, for example a dextran matrix (U.S. Pat. No.5,980,888, herein incorporated by reference). However, a preferredembodiment of the invention encompasses the step of applying cellulosicmaterial comprising a plasmid encoding at least one protein of interestto the wound (e.g., skin wound).

The terms “sample” and “specimen” in the present specification andclaims are used in their broadest sense. On the one hand, they are meantto include a specimen or culture. On the other hand, they are meant toinclude both biological and environmental samples. These termsencompasses all types of samples obtained from humans and other animals,including but not limited to, body fluids such as urine, blood, fecalmatter, cerebrospinal fluid (CSF), semen, saliva, and wound exudates, aswell as solid tissue. However, these examples are not to be construed aslimiting the sample types applicable to the present invention.

DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions useful inlocalized transfer of genetic material or proteins. Moreover, thepresent invention provides methods and compositions for improving and/orcontrolling wound healing by applying a wound care device comprisingHoxD3 and/or HoxA3 and/or HoxB3. In addition, the present inventionprovides methods and compositions for improved wound healing in subjectshaving impaired healing capabilities, such as diabetic subjects.

I. Overview of Skin and External Wound Healing

Skin is a highly versatile organ that serves as a self-renewing andself-repairing interface between the vertebrate body and theenvironment. The skin covers the entire external surface of the body. Inhumans, this includes the external auditory meatus, the lateral aspectof the tympanic membrane, and the vestibule of the nose. The skin iscontinuous with, but distinct from, the mucosae of the alimentary,respiratory, and urogenital tracts, as specialized skin cells at themucocutaneous junctions connects the skin and the mucosae. In additionto its protective functions, skin is capable of absorption, andexcretion, and is also an important primary site of immunosurveillanceagainst the entry of antigens and initiation of the primary immuneresponse. Skin also performs many biochemical synthetic processes thathave both local and systemic effects, and in this sense can be regardedas an endocrine organ.

There are two major types of skin—the thin, hairy (hirsute) skin (whichcovers most of the body), and thick, hairless (glabrous) skin (whichforms the surfaces of the palms of the hands, soles of the feet, andflexor surfaces of the digits). Both types of skin are composed of threebasic layers, namely, the epidermis, the dermis, and the hypodermis. Theprimary differences in the two types of skin are in thickness of theirepidermal and dermal components, and in the presence of hairs with theirattendant sebaceous glands and arrector pili muscles (pilobaceousunits).

The epidermis, a stratified keratinous squamous epithelium primarilycomposed of keratinocytes, can be further divided into several strata(from deep to superficial), namely the stratum basale, stratum spinosum,stratum granulosum, stratum lucidum (where present), and stratum comeum.Epidermal appendages (e.g., pilosebaceous units, sudoriferous gland, andnails) are formed by ingrowth or other modification of the generalepidermis, often referred to as the interfollicular epidermis. Inaddition to keratinocytes, the mature epidermis also contains variousother cells, including melanocytes (i.e., pigment-forming cells),Langerhans cells (i.e., immunocompetent antigen-presenting cells derivedform bone marrow), and lymphocytes. The epidermis also includes Merkelcells, which are thought to be modified keratinocytes.

The population of keratinocytes undergoes continuous renewal, with amitotic layer of cells at the base of the epidermis replacing those shedat the surface. In order to maintain a constant thickness, the rate ofcell production must equal the rate of cell loss. Thus, at any one timein the basal layer of the epidermis there are a variety of keratinocytesin different states of differentiation. These keratinocytes can beclassified into three types according to their clonal proliferativecapacity: 1) stem cells, which have extensive growth capacity; 2)differentiated paraclones, which have limited growth capacity; and 3)intermediate meroclones, which are thought to constitute long-livedprogenitor cells (Trainer et al., 1997 Hum. Mol. Genet. 6:1761–7 [1997];and Barrandon et al., Proc. Natl. Acad. Sci. USA 84:2302–6 [1987]).

When a wound occurs to the skin, the cells must work to close the breachand re-establish the barrier to the environment. The process of woundhealing typically consists of three phases during which the injuredtissue is repaired, regenerated, and new tissue is reorganized into ascar. These three phases can be classified as: a) an inflammation phasewhich begins from day 0 to 3 days; b) a cellular proliferation phasefrom 3 to 12 days; and c) a remodeling phase from 3 days to about 6months.

In the inflammation phase, inflammatory cells, mostly neutrophils, enterthe site of the wound followed by lymphocytes, monocytes, and latermacrophages. Stimulated neutrophils release proteases and reactiveoxygen species into the surrounding medium, with potential adverseeffects on both the adjacent tissues and the invading microorganisms.

The proliferative phase consists of laying down new granulation tissue,and the formation of new blood vessels in the injured area. Fibroblasts,endothelial cells, and epithelial cells migrate in the wound site. Thesefibroblasts produce the collagen that is necessary for wound repair.

In re-epithelialization, epithelial cells migrate from the free edges ofthe tissue across the wound. This event is succeeded by theproliferation of epithelial cells at the periphery of the wound. Ingeneral, re-epithelialization is enhanced by the presence of occlusivewound dressings which maintain a moisture barrier.

Remodeling, the final phase of wound healing, is effected by both thereplacement of granulation tissue with collagen and elastin fibers andthe devascularization of the granulation tissue. Eventually, in mostcases, a scar forms over the wounded area.

II. Gene Transfer and Wound Healing

The present invention provides generally applicable methods andcompositions for improving and/or controlling wound healing, as well asthe transfer of genetic material to a localized area. In someembodiments of the invention, at least one nucleic acid, which can beused in accordance with the invention is contained in an expressioncassette in a vector, preferably in a vector which is applicable in genetherapy. The invention also comprises the use of a vector expressing afusion protein useable according to the invention. The vector which isapplicable in gene therapy preferably contains tissue-specific,wound-specific, skin-specific, cell cycle-specific, cell type-specific,metabolism-specific or constitutively active regulatory sequences whichare functionally linked to the previously described nucleic acid.

The expression vectors used for preparing a polypeptide, which can beused in accordance with the invention, can be prokaryotic or eukaryotic.Examples of prokaryotic expression vectors are the pGEM vectors or pUCderivatives, which are used for expression in E. coli. Examples ofeukaryotic expression vectors are the vectors p426Met25 and p426GAL1(Mumberg et al., Nucl. Acids Res., 22, 5767–5768 [1994]), which are usedfor expression in Saccharomyces cerevisiae, the baculovirus vectors, asdisclosed in EP-B1-0 127 839 or EP-B 1-0 549 721, which are used forexpression in insect cells, and the vectors Rc/CMV, Rc/RSV, and SV40,which are used for expression in mammalian cells. These and additionalsuitable vectors are widely available.

In general, the expression vectors also contain promoters which aresuitable for the respective host cell, such as the trp promoter forexpression in E. coli (See, e.g., EP-BI-0 154 133), the Met 25, GAL 1 orADH2 promoters for expression in yeast (Russel et al., J. Biol. Chem.258, 2674–2682 [1983]; and Mumberg, supra), and the baculoviruspolyhedrin promoter for expression in insect cells (See, e.g., EP-B 1-0127 839). Promoters that permit constitutive, regulatable,tissue-specific, cell type-specific, cell cycle-specific ormetabolism-specific expression in eukaryotic cells are suitable, forexpression in mammalian cells. Regulatable elements in accordance withthe present invention include but are not limited to promoters,activator sequences, enhancers, silencers and/or repressor sequences.

Examples of suitable regulatable elements which permit constitutiveexpression in eukaryotes are promoters which are recognized by RNApolymerase III or viral promoters or enhancers such as the CMV enhancer,CMV promoter (See, e.g., Example 6 and 7), SV40 promoter, LTR promoters(e.g., MMTV-derived as described by Lee et al., Nature 214, 228–232[1981]), and other viral promoter and activator sequences which arederived from, for example, HBV, HCV, HSV, HPV, EBV, HTLV or HIV.Conversely, examples of regulatable elements that permit inducibleexpression in eukaryotes are the tetracycline operator in combinationwith an appropriate repressor (Gossen et al., Curr. Opin. Biotechnol. 5,516–20 [1994]).

The expression of nucleic acids that can be used in accordance with thepresent invention preferably takes place under the control oftissue-specific promoters, with skin-specific promoters, such as thehuman K10 promoter (Bailleul et al., Cell 62: 697–708 [1990]), the humanK14 promoter (Vassar et al., Proc. Natl. Acad. Sci. USA 86: 1563–67[1989]) or the bovine cytokeratin IV promoter (Fuchs et al., The Biologyof Wool and Hair (eds. Rogers et al.) Chapman and Hall, London/N.Y., pp.287–309 [1988]) being particularly preferred. Other examples ofregulatable elements that permit tissue-specific expression ineukaryotes are promoters or activator sequences from promoters orenhancers of genes, which encode proteins that are only expressed inparticular cell types.

Additionally, examples of regulatable elements that permit cellcycle-specific expression in eukaryotes are promoters of the followinggenes: cdc25A, cyclin A, cyclin E, cdc2, E2F, B-myb and DHFR (Zwickerand Müller, Trends Genet. 13, 3–6 [1997]). Examples of regulatableelements that permit metabolism-specific expression in eukaryotes arepromoters that are regulated by hypoxia, by glucose deficiency, byphosphate concentration or by heat shock. An example of a regulatableelement which permits keratinocyte-specific expression in skin is theFiRE element (Jaakkola et al., Gen. Ther., 7: 1640–1647 [2000]). TheFiRE element is an AP-1-driven, FGF-inducible response element of thesyndecan-1 gene (Jaakkola et al., FASEB J., 12: 959–9 [1998]). Examplesfor regulatable elements that allow spatial and temporal expression arenucleic acids coding for a fusion between the site-specific recombinaseCre and a modified estrogen receptor. The expression of this fusionprotein is controlled by a tissue-specific promoter. The resultingcytoplasmic fusion protein can translocate into the nucleus uponadministration of the estrogen analogue tamoxifen and inducerecombination (Feil et al., Proc Natl Acad Sci 93: 10887–90 [1996]).

In order to permit the nucleic acids which can be used in accordancewith the present invention to be introduced into a eukaryotic orprokaryotic cell by means of transfection, transformation or infection,and thereby enabling the polypeptide to be expressed, the nucleic acidcan be present as part of a plasmid, or as a part of a viral ornon-viral vector. Particularly suitable viral vectors in this contextare: baculoviruses, vaccinia viruses, adenoviruses, adeno-associatedviruses and herpesviruses. Particularly suitable non-viral vectors inthis context are: liposomes, virosomes, cationic lipids andpolylysine-conjugated DNA. Examples of viral vectors that are applicablein gene therapy include but are not limited to adenoviral vectors orretroviral vectors (Lindemann et al., Mol. Med. 3: 466–76 [1997];Springer et al., Mol. Cell. 2: 549–58 [1998]).

Eukaryotic expression vectors are suitable for use in gene therapy whenpresent in isolated form, since naked DNA can penetrate into skin cellswhen applied topically (Hengge et al., J. Clin. Invest. 97: 2911–6[1996]; and Yu et al., J. Invest. Dermatol. 112: 370–5 [1999]). Vectorswhich are applicable in gene therapy can also be obtained by complexingthe nucleic acid which can be used in accordance with the presentinvention with liposomes, since this makes it possible to achieve a veryhigh efficiency of transfection, particularly of skin cells (Alexanderand Akhurst, Hum. Mol. Genet. 4: 2279–85 [1995]).

In lipofection, small, unilamellar vesicles consisting of cationiclipids are prepared by subjecting the liposome suspension toultrasonication. The DNA is bound ionically on the surface of theliposomes, specifically in a relationship, which is such that a positivenet charge remains and 100% of the plasmid DNA is complexed by theliposomes. In addition to the DOTMA(1,2-dioleoyloxypropyl-3-trimethylammonium bromide) and DPOE(dioleoylphosphatidylethanolamine) lipid mixtures originally utilizedfor this purpose (Felgner et al., Proc Natl Acad Sci U S A. 84:7413–7[1987]), a large number of new lipid formulations have by now beensynthesized and tested for their efficiency in the transfection ofvarious cell lines (Behr et al., Proc. Natl. Acad. Sci. USA 86,6982–6986 [1989]; Felgner et al., (1994) J. Biol. Chem. 269, 2550–2561[1994]; Gao and Huang, Biochim. Biophys. Acta 1189, 195–203 [1991]).Examples of the new lipid formulations are DOTAPN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium ethyl sulfate orDOGS (TRANSFECTAM; dioctadecylamido-glycylspermine). The Cytofectin GS2888 cationic lipids have also proved to be very well suited fortransfecting keratinocytes in vitro and in vivo (U.S. Pat. No.5,777,153; and Lewis et al., Proc. Natl. Acad. Sci. USA, 93: 3176–3181[1996]). Auxiliary substances which increase the transfer of nucleicacids into the cell can, for example, comprise proteins or peptideswhich are bound to DNA or synthetic peptide-DNA molecules which make itpossible to transport the nucleic acid into the nucleus of the cell(Schwartz et al., Gene Therapy 6, 282 [1999]; and Brandén et al., NatureBiotech. 17, 784 [1999]). Auxiliary substances also encompass moleculeswhich enable nucleic acids to be released into the cytoplasm of the cell(Planck et al., J. Biol. Chem. 269, 12918 [1994]; Kichler et al.,Bioconj. Chem. 8, 213 [1997]).

Liposomes are a pharmaceutically acceptable carrier within the meaningof the present invention. Liposomes comprise multilamellar vesicles(MLVs), small unilamellar vesicles (SUVs) and large unilamellar vesicles(LUVs). Methods for preparing liposome-nucleic acid complexes are knownto the skilled person (e.g., Straubinger et al., Methods of Immunology,101: 512–527 [1983]; and Szoka et al., Proc. Natl. Acad. Sci. USA, 75:4194–4198 [1978]). The term “liposomes” encompasses, for example,liposomal compositions which are disclosed in U.S. Pat. No. 5,422,120,WO 95/13796, WO 94/23697, WO 91/14445 and EP 524,968 B1. Liposomes canbe used with nucleic acids according to the present invention, as wellas for polypeptides according to the present invention or for both as apharmaceutical carrier. Preferably liposomes are used as pharmaceuticalcarriers for the nucleic acids of the present invention. Atherapeutically active substance can also be conjugated to the liposomeor it can be conjugated to a hydrogel polymer, wherein the hydrogelpolymer (or a component of the hydrogel polymer) is conjugated to aliposome or can be enclosed by a liposome. Another especially suitableform vector can be obtained by applying the nucleic acid usableaccording to the invention to gold particles and applying thesetopically with the aid of the so called “gene gun” by shooting them intothe skin or cells (See, e.g., Wang et al., J. Invest. Dermatol., 112:775–81 [1999], and Tuting et al., J. Invest. Dermatol., 111: 183–8[1998]). Devices for performing intradermal injection using pressurehave also been disclosed (See, e.g., U.S. Pat. No. 5,630,796).

For the use of the previously described nucleic acid in gene therapy itis also advantageous if the part of the nucleic acid which encodes thepolypeptide contains one or more non-coding sequences, including intronsequences, preferably between the promoter and the start codon for thepolypeptide and/or a polyA sequence, in particular the naturallyoccurring polyA sequence or an SV40 virus polyA sequence at the 3′ endof the gene since this thereby makes it possible to stabilize the mRNA(Jackson, Cell 74, 9–14 [1993]; and Palmiter et al., Proc. Natl. Acad.Sci. USA 88, 478–482 [1991]).

However, in some particular preferred embodiments, the present inventionprovides methods and compositions involving the use of cellulosicmaterials and plasmid DNA to transfer genetic material to a localizedsite of interest. In particularly preferred embodiments, methylcellulosepellets or films are used as a supporting matrix for plasmid DNA to betransferred to cells surrounding the pellet. In some embodiments, themethylcellulose has limited hydration capabilities, which allows theplasmid DNA to be released from the matrix in a controlled manner overtime and prevents the wound area from becoming overly wet. In otherembodiments, alternative matrix materials are used. However, preferredmatrices all have the ability to immobilize the plasmid DNA to alocalized area and allow the release of the DNA from the matrix to thecells over time. Indeed, during the development of the present inventionsurgical sponges were tested for their suitability for the transfer ofgenetic information, as were naked plasmid DNA constructs (e.g., withoutany supporting matrix). As indicated in Example 9, these experimentswere unsuccessful. Although an understanding of the mechanism is notnecessary in order to use the present invention, it is believed that theuse of surgical sponges resulted in a treatment site that was toohydrated, leading to a more rapid release of the plasmid DNA from thematrix and decreasing the contact of the plasmid DNA with the cells atthe treatment site. For naked DNA, it is believed that the conditions atthe wound site were sufficiently hostile, such that the plasmids werenot efficiently taken-up by the cells. Thus, a matrix such as acellulosic composition (e.g., methylcellulose) is preferred in treatmentof localized areas as the genetic material is efficiently taken up bythe cells, resulting in the expression of the gene of interest. In aparticularly preferred embodiment, the cellulosic material iscarboxymethylcellulose.

In the compositions and methods exemplified herein, pellets containingvarious concentrations of plasmid DNA were used. For example, in someexperiments, 25 μg of plasmid DNA were used, while in other experiments,50 μg of plasmid DNA were used. In preferred embodiments, pelletscontaining 25 μg of plasmid DNA are prepared and then used either alone(i.e., one pellet per wound or area to be treated) or in multiples(i.e., two or more pellets per wound or area to be treated). The numberof pellets used is dependent upon the size of the wound or area to betreated, and upon the stage of wound healing and the desired results.For example, a larger number of pellets may be applied at the onset oftreatment, some or all of which are then replaced as treatmentcontinues, with a lesser or greater number of pellets as needed. Theplasmid DNA contained in these pellets may be the same or different, andmay be at the same or different concentration levels. For example, whendetectable plasmid DNA levels have decreased (typically, at about 4 daysthe plasmid DNA is no longer detectable), “fresh” pellets may be appliedto the treatment area as needed. In other embodiments, when thedetectable protein levels have decreased (typically at about 7 days theprotein expressed by the gene is no longer detectable), “fresh” pelletsmay be applied to the area as needed.

Any plasmid DNA of interest finds use in the methods and compositions ofthe present invention. Indeed, various plasmid constructs were usedduring the development of the present invention, including DNA withreporter (or “signal”) capabilities, as well as DNA that encodesproteins associated with wound healing, and other functions. The dose ofplasmid DNA can be administered on an as-needed basis. Typically, thedose is approximately 1 μg DNA per cm² to be treated. However, doseresponse curves are generated as known in the art on an as needed and/ordesired basis, in order to determine the optimum plasmid concentrationfor use in any particular situation.

The present invention finds use in a number of settings. As exemplifiedherein, one particularly preferred embodiment involves the use of thepresent invention to improve wound healing in subjects with impairednatural healing capabilities, such as diabetic subjects. Nonetheless,the present invention finds use in controlling the rate of wound healing(e.g., by modifying the dose of plasmid DNA). Indeed, the presentinvention finds use in delaying or prolonging wound healing, as well asin accelerating and/or improving wound healing. For example, delayingwound healing is desirable in subjects that tend to experience morefibrosis than observed in most subjects. Thus, the present inventionprovides means to control the rate of wound healing to maximize thetherapeutic benefit for individual patients.

The present invention also finds use in transfer of genetic informationcorresponding to one or more genes. Thus, in some settings, one geneproduct is produced by the subject's cells after the transfer of geneticmaterial from the composition of the present invention, while in othersettings multiple gene products are produced by the subject's cells. Forexample, in one embodiment, the gene for HoxA3 is used in conjunctionwith the gene for HoxD3. As these genes have different functions andeffects, the combination maximizes wound healing in suitable cases. Inalternative settings, the subject is treated with multiple genes inseries. In this case, one gene is used for a suitable length of time andthen another gene is used for another suitable length of time (e.g.,HoxD3 DNA is used first, followed by HoxA3 DNA and/or a gene encoding ananti-fibrotic). In particularly preferred embodiments the subject istreated with one or more of HoxD3 DNA, HoxA3 DNA, and HoxB3 DNA. Thus,the present invention provides maximum flexibility in the treatmentregimen and facilitates optimization of treatment for each subject on anindividualized (i.e., case-by-case) basis.

In addition to improving wound healing on external surfaces such as theskin and mucous membranes, the present invention finds use in improvingthe healing of internal lesions. For example, the present inventionfinds use in improving wound healing associated with surgical incisionsand other localized injury to internal tissues.

Furthermore, the present invention finds use in settings such asinhibition of angiogenesis in tumors. For example, in some embodiments,inhibitory genes are provided that inhibit or prevent blood vesselformation. The compositions of the present invention are placed near orwithin the tumor area, which results in the release of the geneticinformation encoding the inhibitory factor and the subsequent expressionof the genetic information by the cells in the area. Thus, the tumordevelopment is inhibited or stopped due to the lack of vascularization.

III. Protein Transfer and Wound Healing

The present invention also provides methods and compositions forimproving and/or controlling wound healing via transfer of a protein orproteins to a localized area. Indeed, in some embodiments, the presentinvention provides methods and compositions involving the use ofcellulosic materials and at least one recombinant protein to transfer atleast one biologically active protein to a localized site of interest.In particularly preferred embodiments, methylcellulose pellets and/orfilms are used as a supporting matrix for the transfer of protein tocells surrounding the pellet. In some preferred embodiments, themethylcellulose has limited hydration capabilities, which allows theprotein to be released from the matrix in a controlled manner over timeand prevents the wound area from becoming overly wet. In otherembodiments, alternative matrix materials are used. However, preferredmatrices have the ability to immobilize the protein to a localized areaand to allow the release of the protein from the matrix to the cellsover time. It is not intended, however, that application of the proteinbe limited to cellulosic materials.

Alternatively, the above-described proteins that can be used in thepresent invention, are produced as fusion proteins, constituting afunctional variant of one of the previously described proteins or afunctional variant only after the fusion moiety has been eliminated.These fusion proteins include, in particular, fusion proteins that havea content of about 1–300 foreign amino acids, preferably about 1–200foreign amino acids, particularly preferably about 1–150 foreign aminoacids, more preferably about 1–100 foreign amino acids, and mostpreferably about 1–50 foreign amino acids. Such foreign amino acidsequences may be prokaryotic peptide sequences that can be derived, forexample, from E. Coli β-galactosidase. Other preferred examples ofpeptide sequences for fusion proteins are peptides that facilitatedetection of the fusion protein; they include but are not limited togreen fluorescent protein or variants thereof. It is also possible toadd on at least one “affinity tag” or “protein tag” for the purpose ofpurifying the previously described proteins. For example, suitableaffinity tags enable the fusion protein to be absorbed with highspecificity and selectivity to a matrix. This attachment step is thenfollowed by stringent washing with suitable buffers without eluting thefusion protein to any significant extent, and specific elution of theabsorbed fusion protein. Examples of the protein tags which are known tothe skilled person include but are not limited to a (His)₆ tag, a Myctag, a FLAG tag, a hemagglutinin tag, a glutathione-S-transferase (GST)tag, a tag consisting of a an intein flanked by an affinitychitin-binding domain, and a maltose-binding protein (MBP) tag. Theseprotein tags can be located N-terminally, C-terminally and/orinternally.

The proteins that can be used in the methods and compositions of thepresent invention can also be prepared synthetically. Thus, the entirepolypeptide, or parts thereof, can, for example, be produced byclassical synthesis techniques (e.g., Merrifield technique). Particularpreference is given to using polypeptides which have been preparedrecombinantly using one of the previously described nucleic acids.Furthermore, proteins of the present invention can be isolated from anorganism or from tissue or cells for use in accordance with the presentinvention. Thus, it is possible, for example, to purify proteins, whichcan be used in the present invention, from human serum (e.g., Abdullahet al., Arch. Biochem. Biophys., 225:306–312 [1983]). Furthermore, it ispossible to prepare cell lines expressing the proteins of the presentinvention. These cell lines can then be used for isolating the proteinsof interest.

In the compositions and methods exemplified herein, pellets containingvarious quantities of the protein of interest are contemplated. Anywherefrom 1 nanogram to 100 micrograms of the protein of interest areincorporated into the methylcellulose pellets of the present invention.One or multiple pellets are used to treat wounds. The number of pelletsused is dependent upon the size of the wound or area to be treated andon the stage of wound healing or desired results. For example, a largernumber of pellets may be applied at the onset of treatment, some or allof which are then replaced as treatment continues, with a lesser orgreater number of pellets as needed. The protein of interest containedin these pellets may be the same or a different protein, and may be atthe same or different concentration levels.

The present invention finds use in a number of settings. As exemplifiedherein, one particularly preferred embodiment involves the use of thepresent invention to improve wound healing in subjects with impairedhealing capabilities, such as diabetic subjects. Nonetheless, thepresent invention finds use in controlling the rate of wound healing(e.g., by modifying the dose of recombinant protein). Indeed, thepresent invention finds use in delaying or prolonging wound healing, aswell as in accelerating and/or improving wound healing. For example,delaying wound healing is desirable in subjects that tend to experiencemore fibrosis than is typical. Thus, the present invention providesmeans to control the rate of wound healing to maximize the therapeuticbenefit for individual patients.

Various proteins of interest find use in the methods and compositions ofthe present invention. Thus, the present invention finds use in transferof one or more proteins. For example, in one embodiment, recombinantHoxA3 protein is used in conjunction with recombinant HoxD3 protein. Asthese proteins have different functions and effects, the combinationmaximizes wound healing in suitable cases. In alternative settings, thesubject is treated with multiple proteins in series. In this case, oneprotein is used for a suitable length of time and then another proteinis used for another suitable length of time (e.g., HoxD3 protein is usedfirst, followed by HoxA3 protein and/or a protein with anti-fibroticactivity). In particularly preferred embodiments the subject is treatedwith one or more of HoxD3 protein, HoxA3 protein, and HoxB3 protein.Thus, the present invention provides maximum flexibility in thetreatment regimen and facilitates optimization of treatment for eachsubject on an individualized (i.e., case-by-case) basis.

In addition to improving wound healing on external surfaces such as theskin and mucous membranes, the present invention finds use in improvingthe healing of internal lesions. For example, the present inventionfinds use in improving wound healing associated with surgical incisionsand other localized injury to internal tissues.

Furthermore, the present invention finds use in settings such asinhibition of angiogenesis in tumors. For example, in some embodiments,inhibitory proteins are provided that suppress or prevent blood vesselformation. The compositions of the present invention are placed near orwithin the tumor area, which results in the release of the inhibitoryprotein to cells in the area. Thus, the tumor development is inhibitedor stopped due to the lack of vascularization.

IV. Impaired Healing in Diabetics and Wound Healing with HoxD3

Homeobox (Hox) genes are master transcription factors that areassociated with morphogenesis and organogenesis during development andmore recently, in adult tissue remodeling (Myers et al., J. Cell. Biol.,148:343–352 [2000]; Boudreau et al., J. Cell. Biol., 139:257–264 [1997];Boudreau et al., Curr. Op. Cell Biol., 10:640–646 [1998]; van Oostveenet al., Leukemia 13:1675–1690 [1999]; and Cillo et al., Exp. Cell Res.,248:1–9 [1999]). Specifically, HoxD3 was shown to be involved in thespecification of the first and second vertebrae as knock out animalsdisplay homeotic transformations in the atlas region (Condie et al.,Development 119: 579–595 [1993]). It is thought that the depletion ofthese structures is due to a failure of proliferation of precursor cellsresponsible for generating these structures. A function of HoxD3 in cellproliferation was also confirmed by the overexpression of HoxD3 in A549cells, which resulted in the expression of metastasis-associated genes(Omatu et al., Hokkaido Igaku Zasshi 74:367–76, 1999). Moreover, HoxD3was also found to promote endothelial cell (EC) migration by regulatingexpression of integrin αvβ3 and the urokinase plasminogen activator,(uPA) serine proteinase (Boudreau et al. [1997] supra). This change inadhesive properties mediates the EC conversion from a resting to aninvasive state. Again the HoxD3 induced change in adhesion propertiesalso stimulates tumor progression as retroviral overexpression of HoxD3in the chick chorioallantoic membrane assay resulted in the formation ofendotheliomas and vascular malformation (Boudreau et al., [1997] supra).

Previous work described the expression of a number of Hox genes in theskin mainly during fetal wound healing (Stelnicki et al., J. Invest.Dermatol., 110:110–115 [1998]; Stelnicki et al., J. Invest. Dermatol.,111: 57–63 [1998]; and Reigeretal., J. Invest. Dermatol., 103:341–346[1994]). It was noted that many Hox genes, including HoxA4 (HoxD3),HoxA5, HoxA7 and HoxB7, are abundantly expressed in basal keratinocytelayer cells and spread throughout the epidermis during development,suggesting again a role in the regulation of cellularproliferation/adhesion in the developing fetal skin. Interestingly, inthe adult human skin, expression of Hox genes is significantlydown-regulated after birth and is restricted to the upper epidermalkeratinocyte layers and is not present in the dermis (Stelnicki et al.,J. Invest. Dermatol., 110: 110–115 [1998]). Further investigation of Hoxexpression in fetal wounds, which heal without scarring, revealed thatPRX-2 and HoxB13 genes were strongly upregulated in fetal, as comparedto adult wounds (Stelnicki et al., J. Invest. Dermatol., 111: 57–63[1998]). However, these studies did not address the expression of Hoxgenes in the vasculature, nor was re-expression of other Hox genes inadult wound healing investigated.

Thus the present invention, discloses for the first time a direct rolefor the Hox genes in wound-induced expression of Hox genes, particularlyHoxD3. In addition, the present invention discloses for the first timethe expression of Hox B3 and HoxD3 genes in adult skin. Moreover, thepresent invention discloses for the first time, expression of Hox genesin the skin in a cell type other than keratinocytes. In particular, Hoxgenes were found to be expressed in vascular endothelial cells withinthe dermis.

The expression of HoxB3 and HoxD3 was evaluated by in situ hybridizationin non-diabetic mouse (both wild-type and non-diabetic littermates) skinsamples taken at 1, 4, 7, and 14 days post-wounding. In controlunwounded skin samples, relatively weak staining of HoxB3 was observedin the epidermis and hair follicles. In addition, HoxB3 was abundantlyexpressed in vascular endothelial cells lining many medium sized andsmaller vessels of arteriole and venous origin. Four days followingwounding, granulation tissue had begun to form and keratinocytes in theepidermis and cells in the hair follicles showed intense staining forHoxB3. It was then noted that expression of HoxB3 persisted in the EC ofmany of the medium and small size vessels. Although HoxB3 expression wasalso observed in newly forming capillaries in the granulation tissue,expression was somewhat reduced as compared to pre-existing vessels.

By seven days after wounding, keratinocytes and cells in the hairfollicle at the wound continued to show strong expression of HoxB3. Manyinflammatory cells and fibroblasts were also observed near the woundsite, which also showed strong expression of HoxB3. The EC ofcapillaries within the wound also maintained expression of HoxB3.However, again, expression was relatively weak compared to pre-existingcapillaries and larger arterioles further away from the wound site.

By 14 days after wounding, expression of HoxB3 remained high inkeratinocytes, hair follicle epithelium and fibroblasts near the wound.Although many small vessels could not be detected in the wound at thistime, significant expression of HoxB3 in EC of capillaries near therepairing wound site was observed.

Thus, specific changes in Hox gene expression in endothelial cellsduring wound repair in normal and healing-impaired mice were observed.Detailed analysis revealed expression of HoxD3 and HoxB3 in activatedendothelial cells in normal wound repair as shown by in situhybridization (See, Example 2). Surprisingly, wound-induced expressionof HoxD3 was found to be markedly lower and delayed in onset inhealing-impaired diabetic animals (See, Example 12). Moreover, theexpression of HoxD3 was also reduced in ulcers from diabetic patients(See, Example 14), as compared to the HoxD3 expression levels observedduring normal wound healing (See, Example 13). Surprisingly, theseexperiments revealed a down-regulation of HoxD3 selectively in woundshaving impaired healing capabilities, such as diabetic wounds ascompared to normally healing wounds. Thus, it is contemplated that HoxD3is essential for normal wound healing. Moreover, a similar pattern andmagnitude of expression of HoxB3 in wild-type and genetically diabeticmice was observed. This indicates that impaired wound healing is notlikely related to insufficient levels of HoxB3, but rather isselectively due to reduced expression of HoxD3.

The ability of HoxD3 to influence expression of type I collagen mRNA wasexamined during development of the present invention. Humanmicrovascular endothelial cells were stably transfected with cDNAplasmids expressing either HoxD3 or HoxB3 or control empty vectors.Northern blot analysis for the Col1A1 mRNA was performed, and showedthat endothelial cells transfected with HoxD3, but not HoxB3 or controlplasmid, contained levels of Col1A1 mRNA that were 2.5 to 3-fold higherthan HoxB3-transfected or control transfected cells respectively (See,Example 5). Thus, HoxD3 is capable of selectively inducing expression oftype I collagen in EC.

As HoxD3 is selectively down-regulated in wounds having impaired healingcapacities, such as diabetic wounds, wounded diabetic mice were treatedwith methylcellulose pellets containing HoxD3 plasmid, to determinewhether gene transfer of HoxD3 accelerates healing in the diabeticanimals. Indeed, the results indicated that restoration of HoxD3 indiabetic wounds improves overall wound healing (See, Example 7, andFIGS. 2 and 3).

In particular, in the experiments described below, diabetic mice wereused to assess the improvement of wound healing associated with restoredexpression of HoxD3. In these experiments, diabetic mice(C57BL/KsJ-db/db) received full thickness wounds (2.5 cm diameter).Methylcellulose pellets containing 25 μg HoxD3 plasmid were applied tothe open wounds of one group and another group received CMV βgal(control; “control DNA”). Wounds were measured weekly until closure. Thetissues were then processed for Northern blotting and histology.Preliminary results indicated that diabetic control wounds closed in 66days (on average), while the HoxD3 treated wounds closed in 52 days (onaverage). The percent difference in wound closure was significant(p<0.05) at days 7, 14, 21, 28, 35, 42, and 49.

The effect of HoxD3 on 0.8 cm wounds created bilaterally on diabetic(db/db) mice was also investigated. Wound biopsies were taken on days 7,10, 14 and 17. In preliminary studies, it was found that by 17 days,five out of six HoxD3-treated wounds and only one out of sixcontrol-treated wounds had closed. Based on Northern blot analysis,addition of HoxD3 plasmid to diabetic wounds was found to significantlyincrease mRNA levels of collagen at 3, 7, and 10 days post-wounding.

Moreover, the existence of a link between HoxD3 expression and type Icollagen level was examined in diabetic animals. Surprisingly, it wasfound that diabetic mice displayed reduced levels of collagen, whichcould be increased upon administration of HoxD3 (See Example 7 and FIG.4). In addition, HoxD3 was also found to increase angiogenesis duringthe wound healing process of diabetic mice (See Example 3, FIG. 5).

The finding that HoxD3 treatment of wounds was beneficial was unexpectedgiven that HoxD3 is not observed to be expressed in adult human skin,and because of the earlier documented relationship between increasedHoxD3 expression and tumorigenesis (Myers et al., J. Cell Biol.,148:343–351 [2000]). In particular, increased HoxD3 expression has beenshown to promote the development of hemangioma-like structures, whichare aberrant structures typically observed as part of benign tumors.Taken together, these results lead away from a therapeutic use of HoxD3.Thus, the surprising finding of reduced expression of HoxD3 in woundshaving impaired healing capacities, such as diabetic wounds, offered theunexpected possibility of using a cancer-associated protein fortherapeutic purposes.

As discussed above, a deficiency in HoxD3 expression results in markedlyreduced type I collagen production in the healing-impaired wounds ofdiabetic animals. However, the methods and compositions provided by thepresent invention restore HoxD3 expression in diabetic wounds, leadingto enhanced type 1 collagen expression, and improved overall woundhealing. Specifically, the present invention provides methods andcompositions for the more rapid healing of wounds by increasing HoxD3expression in vascular endothelial cells and fibroblasts followingwounding.

Interestingly, homeobox proteins have the ability to enter into cells inthe absence of endocytosis and have the ability to exit cells in theabsence of a signal sequence (See, Prochiantz, Curr Opin Cell Biol12:400–406 [2000]). Once inside the cell, Hox proteins gain access tothe nucleus whereby they regulate gene transcription. Proteins withthese features are termed translocating proteins or messenger proteins.Residues in the third alpha helix of Hox proteins are required forintemalisation (Le Roux et al., Proc. Natl. Acad. Sci. USA, 88:1864–1868[1991]; and Derossi et al., J. Biol. Chem., 269:10444–10450 [1994]).Thus, the methods and compositions disclosed herein differ substantiallyfrom the prior art, as they do not require additional components (e.g.,viruses) for distribution of a transcription factor such as HoxD3protein to the nuclei of cells within a wound. The unexpected use oftranscription factors in wound healing therapy offers the possibility ofa completely new type of therapy. Previously, the only proteins used toimprove diabetic wound repair were soluble secreted growth factors(e.g., PDGF, bFGF, etc.), which triggered several signalling pathwaysand multiple downstream effects. In contrast, the HoxD3 protein of thepresent invention is a nuclear transcription factor, and as such is asignalling cascade end point. Thus, the methods and compositions of thepresent invention comprising HoxD3, are contemplated to provide a moreprecise therapeutic tool. In sum, by increasing levels of HoxD3 inwounds having impaired healing capabilities (e.g., diabetic wounds),through the administration of genetic information or protein, thepresent invention provides the means to promote angiogenesis andcollagen synthesis in wounds having impaired healing capacities.

V. HoxA3 and Wound Healing

The Hox genes in group 3, HoxA3, HoxB3, and HoxD3, exhibit nearlyidentical expression patters and possess approximately 50% identity inprotein coding sequences. Nonetheless targeted inactivation studies havedemonstrated that HoxA3 and HoxD3 have unique functions in vivo.Specifically, mice lacking HoxA3 die after birth with deficiencies inpharyngeal tissues derived from the mesenchymal neural crest (Chisakaand Capecchi, Nature, 350:473–479 [1991]), while mice lacking HoxD3survive despite having malformations in the somitic,mesodermally-derived tissues of the axial skeleton (Condie and Capecchi,Dev. Biol., 119:579–595 [1993]). The uniqueness of the single mutantphenotypes suggested that HoxA3 and HoxD3 have qualitatively differentfunctions. However, the observation that vertebral defects in HoxD3mutant mice were exacerbated by removing HoxA3 function, suggested thatthere is also a functional overlap between these genes and thus, aquantitative aspect to their combined activities (Condie and Capecchi,[1993] supra; and Manley and Capecchi, Dev. Biol., 195:1–15 [1998]).

In fact, during embryogenesis HoxD3 and HoxA3 were shown to befunctionally interchangeable if expressed within the proper context.Specifically, when HoxD3 was expressed from the HoxA3 locus of HoxA3null mice, HoxD3 was capable of rescuing the otherwise lethal HoxA3 nullphenotype (Greer et al., Nature, 403:661–664 [2000], herein incorporatedby reference). However, it was not clear whether these two Hox geneswould also be equivalent in adult tissues. Studies of compound mutantsof Hox9 genes revealed markedly different effects on embryonic and adultmammary gland development indicating that Hox genes may perform multipleroles that change with time (Chen and Capecchi, Proc. Natl. Acad. Sci.,96:541–546 [1999]).

With this in mind, the influence of HoxA3 in adult microvascularendothelial cells was examined during the development of the presentinvention. As described in more detail below in Examples 15–23, HoxA3has been shown for the first time to promote EC migration in culture andangiogenesis in vivo. The present studies also indicate that in EC, theHoxA3 gene may cooperate with the paralogous HoxD3 gene to promote ECmigration. In particular, HoxD3 was shown to induce expression of thelatent MMP-2 pro-enzyme, while HoxA3 induces expression of MMP-14.MMP-14 not only activates latent MMP-2, but can also directly functionas a potent matrix degrading enzyme (See, Werb, Cell, 91:439–442[1997]). In addition, HoxA3 upregulates expression of uPAR, the receptorfor uPA, whose expression is induced by HoxD3 (Boudreau et al., J. CellBiol., 139:257–264 [1997]). Thus, during development of the presentinvention, HoxA3 and HoxD3 have been shown to mediate expression ofdistinct, yet functionally related genes, which can act in a cooperativemanner to promote EC migration. Nonetheless, an understanding of themechanism(s) is not necessary in order to make and use the presentinvention.

Several reports have emphasized additional interactions between uPAR,integrins and MMPs. For instance, uPAR can directly ligate the αvβ3integrin to enhance cell-cell interactions (Tarui et al., J. Biol.Chem., 276:3983–90 [2000]). The αvβ3 integrin can also bind and localizeactive MMP-14 and MMP-2 on invading cells (Brooks et al., Cell,92:391–400 [1998]; and Hofiann et al., Int. J. Cancer, 87:12–19 [2000]),while MMP-14 is able to modify the β3 integrin subunit resulting in itsactivation and enhanced ability to bind MMP-2 (Deryugina et al., Int. J.Cancer, 86:15–23 [2000]). Thus, when the paralogous HoxA3 and HoxD3genes or gene products are applied together to a wound, they areexpected to act synergistically to induce an angiogenic phenotype in EC.Nonetheless, an understanding of the mechanism(s) is not necessary inorder to make and use the present invention.

It is also worth noting however, that HoxA3 infrequently generateshemangioma-like lesions. Recent studies investigating components of theplasminogen/plasminogen activator system elegantly demonstrated the needfor tight regulation of proteolysis in order for newly formed vessels tostabilize and mature (Bajou et al., J. Cell Biol., 152:777–784 [2001]).Since HoxA3 targets uPAR and MMP-14, and can act to localize andregulate serine or metallo-proteinase activity in endothelial cells,HoxA3 is contemplated to prevent diffuse proteolysis, which if leftunchecked, could lead to the formation of irregular and cavernous cysticstructures characteristic of hemangiomas (Werb, Cell, 91:439–442[1997]); Ossowski and Aguire-Ghiso, Curr. Opin. Cell Biol., 12:613–620[2000]; Montesano et al., Cell, 62:435–445 [1990]; and Takahashi et al.,J. Clin. Invest., 93:2357–2364 [1994]). Nonetheless, an understanding ofthe mechanism(s) is not necessary in order to make and use the presentinvention.

Differences in EC behaviour induced by HoxA3 and HoxD3 were alsoapparent in tissue culture studies disclosed herein. As shown in FIG. 9,HoxA3-transfected EC possess a greater ability to migrate intothree-dimensional fibrin gels, whereas HoxD3-transfected EC possess agreater ability to migrate on fibrinogen. The findings thatHoxA3-induced migration was impaired in the presence of functionblocking antibodies against uPAR are consistent with previous reportswhich demonstrated that while migration into a pure fibrin matrix wasnot effected by antibodies against αvβ3, antibodies against uPAR couldabolish invasion (Kroon et al., Am. J. Pathol., 154:1731–1742 [1999]).Thus, some embodiments of the present invention comprise both HoxA3 anduPAR function blocking antibodies when it is desirable to utilize somebut not all of the HoxA3-induced functions. Furthermore, although theMMP system may also function to mediate migration in fibrin-richmatrices in the absence of the plasminogen activator system (Hiroaka etal., Cell, 95:365–377 [1998]), addition of the MMP inhibitor GM6001 hadno impact on HoxA3-depdendent migration the fibrin system describedherein. Nonetheless, an understanding of the mechanism(s) is notnecessary in order to make and use the present invention.

VI. Selection of Compounds Capable of Modulating HoxA3 Activity

The present invention also provides novel tools and techniques foridentifying compounds capable of enhancing or inhibiting various HoxA3activities associated with wound repair. For instance, the ability of acandidate compound to modulate HoxA3-induced angiogenesis can beassessed by observing blood vessel formation in CAM grafted with controltumors or HoxA3-transfected tumors, in the presence and absence of thecandidate compound. Alternatively, the ability of a candidate compoundto modulate the HoxA3-induced expression of uPAR or MMP14 can beassessed by comparing uPAR or MMP14 expression levels in controlepithelial cells and in HoxA3-transfectants, in the presence and absenceof the candidate compound. In addition, the ability of a candidatecompound to modulate HoxA3-induced migration of epithelial cells throughfibrin or on fibrinogen can be assessed by observing the migration ofcontrol epithelial cells and HoxA3-transfectants, in the presence andabsence of the candidate compound. Moreover, the ability of a candidatecompound to modulate HoxA3-induced migration of keratinocytes on plasticcan be assessed by observing the migration of control keratinocytes andHoxA3-transfectants, in the presence and absence of the candidatecompound.

The inventors contemplate many other means of screening compounds. Theexamples provided above are presented merely to illustrate a range oftechniques available. One of ordinary skill in the art will appreciatethat many other screening methods can be used successfully.

VII. HoxB3 and Wound Healing

As shown herein for the first time, treatment of diabetic wounds withHoxB3 DNA alone significantly enhanced the rate of wound closure, ascompared to control DNA-treated diabetic wounds (See, FIG. 14). Thisfinding was unexpected given that the HoxB3 expression level observed indiabetic wounds was on the order of that observed in the wounds ofnon-diabetic subjects. This was also surprising given that transfectionof human microvascular endothelial cells with HoxB3 did not result in anincrease in type I collagen expression. Thus, although HoxB3, HoxA3 andHoxD3 apparently play distinct roles in the process of wound healing,all are effective as therapeutic agents for the treatment of diabeticwounds. Furthermore, it is contemplated that wound care devicescomprising two or more of HoxB3, HoxA3 and HoxD3 provide even morepotent treatments.

VIII. Selection of Compounds Capable of Modulating HoxB3 Activity

The present invention also provides novel methods and compositions foridentifying compounds that enhance or inhibit various HoxB3 activitiesassociated with wound repair. For instance, the ability of a candidatecompound to modulate HoxB3-enhanced wound closure can be assessed byobserving the closure of HoxB3 protein or DNA-treated 2.5 cm wounds onthe backs of diabetic (db/db) mice, in the presence and absence of thecandidate compound. Alternatively, the ability of a candidate compoundto modulate the HoxB3-induced capillary morphogenesis can be assessed byobserving capillary morphogenesis of HoxB3-tranfected HMEC-1 cellscultured on reconstituted basement membrane, in the presence and absenceof a candidate compound. In addition, the ability of a candidatecompound to modulate the HoxB3-induced expression of Ephrin A1 can beassessed by comparing Ephrin A1 expression levels in control epithelialcells and in HoxB3-transfectants, in the presence and absence of thecandidate compound. The present invention provides many other means ofscreening compounds. The examples provided above are presented merely toillustrate a range of techniques available. One of ordinary skill in theart will appreciate that many other screening methods can be usedsuccessfully.

Experimental

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); mM (millimolar); μM(micromolar); N (Normal); mol (moles); mmol (millimoles); μmol(micromoles); nmol (nanomoles); g (grams); mg (milligrams); μg(micrograms); ng (nanograms); 1 or L (liters); ml (milliliters); μl(microliters); cc (cubic centimeters); cm (centimeters); mm(millimeters); μm (micrometers); run (nanometers); ° C. (degreesCentigrade); bp (base pair); kb (kilobase); PCR (polymerase chainreaction); CMV (cytomegalovirus); βgal (beta-galactosidase); cpm (countsper minute); BrdU (bromodeoxyuridine); SDS (sodium dodecyl sulfate);MgCl₂ (magnesium chloride); NaCl (sodium chloride); EDTA (ethylenediamine tetraacetic acid); DEPC (diethyl pyrocarbonate); SSC (saltsodium citrate); BSA (bovine serum albumin); FCS (fetal calf serum); PBS(phosphate buffered saline); Tris (tris(hydroxymethyl)aminomethane); H₂O(water); IgG (immunoglobulin); Clonetics (Palo Alto Calif.); Invitrogen(Invitrogen Life Technologies, Carlsbad, Calif.); Roche (F. Hoffmann-LaRoche Limited, Basel, Switerland); Sigma (Sigma Chemical Co., St. Louis,Mo.); Jackson (Jackson Laboratory, Bar Harbor, Me.); Boehringer Mannheim(Boehringer Mannheim, Indianapolis, Ind.); Fisher (Fisher Scientific,Pittsburgh, Pa.); Ambion (Ambion, Austin, Tex.); Vector (VectorLaboratories, Burlingame, Calif.); Zymed (Zymed Laboratories, South SanFrancisco, Calif.); Pharmingen (Pharmingen, San Diego, Calif.); Qiagen(Qiagen, Valencia, Calif.); Life Technologies (Life Technologies,Rockville, Md.); Alpha Innotec (Alpha Innotec, San Leandro, Calif.); andATCC (American Type Culture Collection, Manassus, Va.).

In some experiments, genetically diabetic C57BL/KsJ-db/db mice, theirnon-diabetic litter mates, and wild-type C57B1/129 mice obtained fromJackson were used. The animals were housed in the University ofCalifornia, San Francisco animal care facility, and all procedures wereapproved by the Committee on Animal Research. All mice were between 8 to12 weeks at time of wounding.

In preferred embodiments and in the experiments described below, themethylcellulose used is carboxymethylcellulose (sodium salt) (Sigma#C-5013). Water soluble polymer and 1% solution at 25° C. has aviscosity of 1500–3000 cps. To prepare the methylcellulose pelletscontaining plasmid DNA, 25 μg of plasmid DNA (in a volume of 25 μl ofwater) was mixed with 25 μl of a 1% solution of methylcellulose(dissolved in sterile water). After brief mixing, 50 μl of this solutionwas dropped in a bacterial culture plate without spreading, and allowedto dry for 1–2 hours at room temperature. The methylcellulose filmformed a spot approximately 1 cm in diameter, which is then peeled offthe culture dish and placed directly onto the recently made open woundor other site to be tested. For example, in experiments involving 0.8 cmwounds, one 50 μl pellet was used, while for 2.5 cm wounds, four or fiveof the 50 μl pellets were placed adjacent to or in the wound. In otherembodiments, HoxD3 protein is used in conjunction with the pellets,while in other embodiments, a retroviral vector is used for genedelivery to a target site of interest.

EXAMPLE 1 Wounding

Prior to wounding, wild-type and diabetic mice were anaesthetized with0.04 cc of ketamine/xylazine (50 mg/cc/2.5 mg/cc). The dorsa of the micewere shaved and a 1.5 cm linear incision was made through the panniculuscamosus and closed with two interrupted 4-0 nylon sutures (e.g., placed0.5 cm apart). The mice were sacrificed at post wound days 1, 4, 7, and14, by anaesthetic overdose and bilateral thoracotomies were performed.To ensure consistent sampling of the wounds, a standard 0.5×0.5 cmsection from the center of the wound was obtained and processed for RNA,in situ hybridization, and/or immunohistochemistry. A total of 16wild-type mice (5 mice at day 0; 3 at days 1, 4, and 7 post-wounding;and 2 at day 14 following wounding) were used and 12 diabetic (db/db)mice (3 mice each at days 0, 4, 7, and 14 days post-wounding) were usedin the analyses described herein.

EXAMPLE 2 In Situ Hybridization

Since diabetic mice have been reported to show a delay in wound repairand angiogenesis, the expression of both HoxB3 and HoxD3 afterproduction of a linear wound in diabetic mice was investigated. In theseexperiments, in situ hybridization for HoxB3 or HoxD3 was performed on aminimum of 8 sections (for each Hox gene) from each of the woundedtissues collected (See, Example 1). Wound tissues were fixed in 10%formalin/phosphate buffer overnight at room temperature. Tissues werethen dehydrated in ethanol and paraffin embedded. Then, 5 μm sectionswere place on Vectabond (Vector)-treated slides. Prior to use, theslides were heated at 80° C. for 30 minutes, deparaffinized in xylene,and rehydrated in a graded ethanol series, as known in the art. Tissueswere post-fixed in 4% paraformaldehyde for 5 minutes at roomtemperature. Tissue sections were then treated with RNAse-freeproteinase K (Ambion) at 20 μg/mL in 10 mM Tris pH 7.5 and 1 mM EDTA,for 10 minutes at 37° C. Sections were again post-fixed with 4%paraformaldehyde for 5 minutes at room temperature, washed anddehydrated through a graded ethanol series, as known in the art.

Tissue sections were prehybridized in 50% formamide, 3 mM NaCl, 10 mMTris pH 7.5, 1 M EDTA, 1% blocking reagent (Boehringer Mannheim), 10%dextran sulfate, and 150 mg/ml tRNA for 1 hour at 45° C. The tissueswere then hybridized with digoxigenin-labeled riboprobes for HoxB3 orHoxD3, in either the sense or anti-sense orientation. These riboprobeswere generated using linearized plasmids and the Genius RNA Labeling kit(Boehringer Mannheim) as known in the art (See, Myers et al., supra; andBoudreau et al. [1997], supra). The riboprobes were diluted inhybridization solution to a concentration of 800 ng/ml, and incubatedwith tissue sections overnight at 45° C. Following hybridization, theslides were washed with 2×SSPE for 5 minutes at room temperature then0.2×SSPE for 1 hour at 50° C. Tissue sections were blocked in 2%blocking reagent (Boehringer Mannheim), 100 mM Tris pH 7.5, and 150 mMNaCl, for 45 minutes at room temperature. Sections were then washed with1% BSA, 0.3% Triton-X100, 100 mM Tris pH 7.5, and 150 mM NaCl, andincubated overnight with a 1:500 dilution of anti-digoxigeninalkaline-phosphatase conjugated antibody (Boehringer Mannheim). Thetissue sections were subsequently incubated in 20 μl/ml nitrobluetetrazolium/5-bromo-4-chloro-3-indoyl phosphate substrate (BoehringerMannheim) until they reached the desired intensity. The reaction wasstopped in Tris/EDTA, pH 8, and sections were counterstained with 1%Fast Green and mounted with an aqueous mounting compound known asCrystal Mount (Fisher).

Thus, HoxB3 and HoxD3 expression in linear wounds of wild-type animalswere examined. As with HoxB3, expression of HoxD3 was observed in bothkeratinocytes and hair follicle epithelial cells in unwounded skin.However, in contrast to HoxB3, no significant levels of expression ofHoxD3 were observed in the resting EC of small vessels in the sub-dermallayer or in capillaries within the dermal layer. However, occasional lowlevels of expression were observed in some small arterioles.

Nonetheless, within 1 day after wounding, an increase in intensity ofHoxD3 expression in keratinocytes, hair follicle epithelium,fibroblasts, and notably, small and medium sized vessels adjacent to thewound site was observed. Furthermore, an increase in expression in HoxD3in capillaries immediately adjacent to the wound site was observed,although there was no increase in HoxD3 expression in capillariesfurther away. The increase in expression of HoxD3 in fibroblasts and ECof vessels near the wound site was maintained at 4 days after wounding.Indeed, high levels of HoxD3 expression were maintained in EC ofcapillaries in the wound tissue through 7 days after wounding. However,by 14 days post-wounding, expression of HoxD3 in EC had begun todecline.

No difference in intensity or localization in either HoxD3 or HoxB3expression was observed in unwounded skin of diabetic mice, as comparedto unwounded skin of wild-type mice. Furthermore, although a decrease inthe extent of new capillaries formed and vessel density was noted indiabetic mice following wounding, no significant changes in intensity orlocalization of HoxB3 expression in these vessels adjacent or away fromthe wounds was observed for up to 14 days after wounding.

Expression of HoxD3 in wounded wild-type and diabetic (db/db) mice wasalso examined. Expression of HoxD3 was observed at four days followingwounding and capillaries near the wounds in diabetic mice expressedrelatively low levels of HoxD3 as compared to wild-type animals. Inaddition, expression in control and diabetic (db/db) mice at 7 daysfollowing wounding was analyzed. Although both tissues showed similarlevels of HoxD3 expression in the epidermis, expression of HoxD3 wasmarkedly reduced in EC of vessels near or within the wounds of diabetic(db/db) mice as compared to wild-type animals. Finally, although the ECof capillaries and fibroblasts within the wounds of wild-type miceexpress HoxD3, capillary EC in wounds of diabetic (db/db) animals showedlittle HoxD3.

EXAMPLE 3 Immunohistochemistry

For immunohistochemistry, 5 μm sections embedded in paraffin weredeparaffinized in 3 washes of Hemo-De (Fisher) for 5 minutes. Sectionswere then rehydrated through a graded ethanol series. Tissue sectionswere then treated with 1 μg/μL of protease K for 10 minutes at 37° C.and then washed under running water for 10 minutes. Sections wereblocked for endogenous peroxidase activity with Peroxoblock (Zymed) for45 seconds at room temperature. Sections were then incubated in 1% goatserum/PBS for 15 minutes at room temperature. Sections were thenincubated with 1:250 dilution of rat-anti-mouse CD31 (PECAM) antibody(Pharmingen) overnight at 4° C. Sections were incubated with a 1:500dilution of biotin-conjugated goat anti-rat IgG (Jackson Laboratories)for 1 hour at room temperature and then incubated for 1 hour at roomtemperature with the Vectastain ABC Reagent (Vector). Slides weredeveloped using the DAB plus kit (Zymed). Sections were dehydratedthrough ethanol and mounted with Permount.

The production of type I collagen has been shown to be decreased inother diabetic wound models (Darby et al., Intl. J. Biochem. Cell Biol.,29:191–200 [1997]; and Bitar and Labbad, J. Surg. Res., 61:113–119[1996]). To confirm that linear wounds in db/db diabetic mice, whichexhibit reduced HoxD3, also show a reduction in type I collagendeposition and expression, the following experiments were conducted.Paraffin-embedded sections of wild-type or diabetic mouse skin weretaken 7 and 14 days after wounding. Trichrome staining showed thatcollagen deposition was indeed reduced in diabetic mice as compared towild-type mice.

FIG. 1 provides photographs of trichrome-stained tissue sections showingcollagen deposition in db/db wounds 14 days after creation of 1 cm openwounds. Panel A provides a low power image of a db/db wound treated withcontrol DNA. As indicated in this Panel, there is limited collagendeposition (shown in blue). Panel B provides a higher power image ofPanel A. Panel C provides a low power image of a HoxD3 treated wound. Asindicated in this Panel, there is more extensive collagen deposition inthis treated wound, as compared to the control. Panel D provides ahigher power image of Panel C, showing extensive collagen deposition andthe presence of small microvessels in the treated wound. Similarly, FIG.5 provides photographs of trichrome-stained tissue sections from db/dbwounds treated with HoxD3 DNA 21 and 42 days post-wounding. These laterimages depict the appearance of collagen fibrils and small blood vesselsin healing wounds treated with HoxD3 DNA.

Furthermore, Northern blot analysis of total RNA taken from wild-type ordiabetic wounds at 7 days, indicated that the decrease in type Icollagen was related to a decrease in expression of Col1A1 mRNA levels.In addition, the burst strengths of the diabetic wounds were less than30% of the wild-type wounds when tested with a tensiometer (Instron,Canton, Mass.).

EXAMPLE 4 Cell Culture and Transfection

An immortalized human dermal microvascular endothelial cell line HMEC-1(Ades et al., J. Invest. Dermatol., 99:683–690 [1992]), kindly providedby T. Lawley of Emory University, Atlanta Ga., was used in theseexperiments. These cells have previously been shown to maintain manyproperties of primary dermal microvascular cells in culture includingthe ability to undergo capillary morphogenesis when cultured on basementmembrane (Matrigel) and to maintain expression of a number ofendothelial cell surface markers (Xu et al., J. Invest. Dermatol.,102:833–837 [1994]). Cells were maintained in media MCBD 131supplemented with 10% FCS, gentamicin and 1% hydrocortisone (Sigma), andpassaged using calcium and magnesium-free PBS supplemented with 0.053 mMEDTA. Cells were transfected with 2 μg DNA using Effectene reagent(Qiagen) and stable transfectants were selected using 50 μg/ml G418.Expression vectors for HoxD3 and HoxB3 were prepared using methods knownin the art (See, Myers et al., supra; Boudreau et al. [1997], supra).

Primary cultures of human dermal microvascular endothelial cells werepurchased from Clonetics. Recombinant human VEGF and bFGF were purchasedfrom R&D Systems, and Matrigel was obtained from Collaborative Research.Endothelial cell culture on Matrigel basement membranes was performed aspreviously described (Boudreau et al., J. Cell. Biol., 139:257–264[1997]). To release cells from Matrigel for protein or mRNA isolation,cultures were suspended in PBS without Ca²⁺ or Mg²⁺, containing 0.5 mMEDTA and incubated on ice for 1 hour to allow the Matrigel to disperse.

EXAMPLE 5 RNA Isolation and Northern Blot Analysis

Mouse wound tissue obtained as described above was excised and snapfrozen in liquid nitrogen and subsequently homogenized prior to RNAisolation using TRIZOL Reagent (Life Technologies). For cultured cells,RNA was isolated with RNA Easy spin columns (Qiagen). For Northern blotanalysis, a total of 5–10 μg (tissue) or 10–20 μg (cells) of total RNAwas electrophoresed through 1% agarose formaldehyde gels using standardmethods. Ribosomal RNA was visualized by staining with 1% ethidiumbromide. ³²PdCTP-labeled probes were prepared using the Decaprime kit(Ambion) and purified using Sephadex G-25 columns (Boehringer Mannheim).The blots in FIG. 4 were probed with 1×10⁶ cpm of labeled cDNA probedirected against human Col1A1 (ATCC), and washed 3 times using lowstringency conditions of 2% SSC/0.1% SDS for 30 minutes at 45° C. formouse tissue, while blots containing human cell derived RNA were subjectto an additional high stringency wash in 0.2% SSC at 65° C. for 30minutes. Similarly, the blots in FIG. 8 were probed with Hybridsol I(Oncor) hybridization buffer containing probes for human uPA, uPAR,MMP-2, β3 integrin or MMP-14. The uPA, uPAR, and MMP-2 cDNAs werepurchased from ATCC, while the cDNA of the β3 integrin was obtained fromD. Cheresh (Scripps). The cDNA probe for human MMP-14 corresponds toGenBank Accession No. NM004995. Membranes were exposed to Kodak BioMaxfilm or Kodak M5 X-Omat film at −70° C. mRNA levels were quantitated byscanning densitometry of the film and normalized to ethidium bromidestaining of total ribosomal RNA using Chemlmager 4000 software (AlphaInnotech).

Northern blot analysis for the Col1A1 mRNA showed that endothelial cellstransfected with HoxD3, but not HoxB3 or control plasmid, had levels ofCol1A1 mRNA which were 2.5 to 3-fold higher than HoxB3-transfected orcontrol-transfected cells respectively (See FIG. 4).

EXAMPLE 6 Construction of HoxD3 Expression Plasmid

In these experiments, construction of the HoxD3 expression plasmid isdescribed. A full-length human HoxD3 clone was isolated using standardPCR, with primers directed against the published sequence (GenBankAccession Number D11117). The DNA sequence of the human HoxD3 clone isprovided as SEQ ID NO:1, while the predicted protein sequence of thehuman HoxD3 clone is provided as SEQ ID NO:2. The forward primer usedhad the sequence 5′-AGG GTC AGC AGG CCC TGG AGC-3′ (SEQ ID NO:3), andthe reverse primer had the sequence 5′-AGA GCG GGG AAG GGG GTT CCC GAACT (SEQ ID NO:4). The 3.4 kb PCR product was inserted into the Topo IIcloning vector (Invitrogen). The 3.4 kb insert was then removed withKpnI/BamHI and inserted into the pcDNA3 expression vector under controlof the CMV promoter (Invitrogen). Plasmid DNA was then purified using aQiagen Maxi-prep kit (Qiagen), per the manufacturer's instructions.

EXAMPLE 7 Gene Transfer of Hox D3

In these experiments, gene transfer of HoxD3 to diabetic mice wasinvestigated. As the experiments described above indicate thatexpression of HoxD3 is decreased in wounds of diabetic (db/db) animals,experiments were conducted using diabetic (db/db) mice withfull-thickness wounds administered as described in Example 1.Methylcellulose pellets containing 25 μg HoxD3 plasmid were applied tothe wounds. Control (diabetic) animals received methylcellulose pelletscontaining CMV βgal as a control plasmid.

The wounds were measured weekly until closure. The tissues were thenprocessed for Northern blotting and histology using the methodsdescribed in the previous Examples. The results indicated that diabeticcontrol wounds closed in 66 days (on average), while the HoxD3 treatedwounds closed in 52 days (on average). The percent of wound closure wassignificant at day 21 (p=0.02), day 14, and day 28 (p=0.06). In theseexperiments, five animals were treated with HoxD3 plasmid, while threeanimals were treated with control DNA.

FIG. 2 provides a graph showing closure of 2.5 cm wounds in diabetic(db/db) mice treated with HoxD3 DNA or control DNA. As indicated in thisFigure, mice treated with HoxD3 DNA (indicated by diamonds in thisgraph) showed a significantly (p=0.02) greater degree of closure at day21, as compared to mice treated with control DNA (indicated by squaresin this graph) treated wounds. The relative difference in closure inHoxD3-treated wounds as compared to control DNA was significant (p=0.05)at 7, 14, 21, 28, 35, 42, and 49 days, as determined using a student's ttest.

The effects of HoxD3 on 0.8 cm wounds created bilaterally on diabetic(db/db) mice were also investigated. Wound biopsies were taken on days7, 10, 14 and 17. In preliminary studies, it was found that by 17 days,5 out of 6 HoxD3 treated wounds and only 1 out of 6 control DNA treatedwounds had closed. Based on Northern blot analysis, addition of HoxD3plasmid to diabetic wounds was found to significantly increase collagenmRNA levels at 3, 7, and 10 days post-wounding. FIG. 3 providesphotographs of two mice at day 7 and day 14 after wounding. Asindicated, the HoxD3-treated wounds showed a significant difference inclosure, as compared to the controls.

FIG. 4 provides Northern blot analyses of type I collagen RNA expressionin control and HoxD3 treated 0.8 cm wounds. Panel A shows expression oftype I collagen (Col1A1) mRNA and corresponding total RNA loading (rRNA)in bilateral wounds from a diabetic (db/db) mouse treated with controlDNA or HoxD3 DNA for 7 days. Panel B shows that expression of type Icollagen mRNA remains higher in tissues taken from HoxD3 treated wounds,as compared to bilateral control DNA-treated wounds from the sameanimals, as described for Panel A, after 10 days. The correspondingribosomal RNA (rRNA) loading controls are shown below. Panel C showsthat expression of type I collagen mRNA remains higher in HoxD3 treatedwounds as compared to corresponding control DNA-treated wounds from thesame animals as described for Panels A and B, after 17 days.

EXAMPLE 8 Wound Closure and HoxD10

In this Example, experiments conducted to determine the effects ofHoxD10 on wound closure are described. Wounds (0.8 cm) were made in wildtype C57B1 mice as described above. The wounds were treated withmethylcellulose pellets containing 25 μg of CMV βgal cDNA (control) orpellets containing 25 μg of HoxD10 cDNA expression vectors. Open woundsize was measured at the time of wounding and at 3, 5, 10 and 11 daysfollowing wounding. Closure of HoxD10 treated wounds was significantlyimpaired (p<0.05) on days 3 and 10, as compared to control treatedwounds (n=3). Thus, HoxD10 was shown to delay wound healing.

The use of HoxD10 inhibitors to enhance wound healing is contemplated.In particular, methylcellulose pellets containing HoxD10 RNAi or controlpellets containing CMV βgal RNAi are prepared. Eight-millimeter woundsare made on the backs of C57B1 mice and the RNAi-containingmethylcellulose pellets are applied to the wounds. Wound size ismeasured at 0, 3, 5, 10, and 15 days after wounding.

EXAMPLE 9 Use of Naked DNA and Sponges to Transfer DNA

In this Example, experiments conducted using DNA without methylcelluloseor another carrier are described. Linear wounds were made on wild-typeC57B1 mice, as described above. In initial experiments, 50 μg of CMVβgal plasmid DNA was directly applied by pipet to freshly made linearwounds. After 24 and 48 hours, 1 cm of tissue located peripheral to theinjection area was harvested. The tissue was placed in 20% sucrose/PBSfor 1 hour, embedded in OCT, and frozen in dry ice and ethanol. Then, 5μm cryosections were prepared and stained for β-galactosidase activityas known in the art (See e.g., Hengge et al., Nat. Genet., 10:161–166[1995]). Using this method, no positive staining for β-galactosidaseactivity was noted.

In subsequent studies, linear wounds were made as described above. Threedays later, mice were injected with 50 μg of CMV βgal plasmid DNA, usinga 30 gauge needle. DNA was administered either within 1 mm from the edgeof the existing wound or alternatively, the wound was reopened in thedermis and the DNA was injected into the wound.

Mice were sacrificed 24 hours following injection. Then, 1 cm of tissueperipheral to the injection area was taken, fixed, and stained asdescribed above. No significant staining was seen using either method ofinjection.

Finally, polyvinyl sponges (i.e., commonly used as surgical sponges)were soaked in a 100 μl solution containing 50 μg of plasmid CMV βgalplasmid DNA as known in the art (See e.g., Thornton et al., Biochem.Biophys, Res. Commun., 246:654–659 [1998]). Linear wounds were made inmice as described above. The prepared sponges were then inserteddirectly under the dermis and secured with a vicryl suture. Wounds wereclosed with 4 sutures. Tissue containing the sponges was harvested at 1,2, 5, and 7 days following wounding/sponge application and processed asdescribed above (See e.g., Hengge et al. supra). Some scattered positivestaining cells (likely fibroblasts) were observed in the day 1 and 2tissues, but this method did not appear to be an effective means totransduce the DNA. Indeed, this Example indicates that the cellulosicmaterial of the preferred embodiments is a much better method than thatdescribed in this Example in which naked DNA and polyvinyl sponges wereused.

EXAMPLE 10 Plasmid DNA Uptake and Expression in Chicks

In this Example, experiments were conducted to observe DNA uptake andexpression in chicks. As described above, CMV βgal plasmid DNA, c-myctagged Hox plasmid DNA, and HA-tagged Hox plasmid DNA was incorporatedinto a 0.5% methylcellulose pellet. The plasmid DNA/methylcellulosemixture was placed onto the chorioallantoic membranes (CAM) of 10 daychick embryos adjacent to pellets containing 50 ng of recombinant VEGF.The CAMs were prepared as described in the art (Brooks et al., Science264:569–571 [1994]). Expression of β-galactosidase was observed inendothelial, fibroblast, and epithelial cells 48 hours after plasmidDNA/methylcellulose pellet deposition. Expression in chick endothelialcells was confirmed by double immunofluorescence against an endogenousendothelial cell specific marker (e.g., Von Willebrand factor), and thec-myc or HA-tagged Hox protein.

EXAMPLE 11 Effects of Recombinant HoxD3 Protein on Cultured Cells

The use of any suitable gene expression system, including prokaryotic,yeast, insect and mammalian cell expression systems, is contemplated forthe production of the recombinant HoxD3 (rHoxD3) protein of theinvention. In a preferred embodiment, the rHoxD3 protein is producedwith a baculovirus expression system, as known in the art (Ausubel etal., (eds.), Current Protocols in Molecular Biology, Chapter 16.9–16.11,John Wiley and Sons Inc., New York [1997]). In one embodiment, therHoxD3 protein is expressed with an influenza hemagglutin (HA) epitopetag. rHoxD3 protein is then purified via high performance liquidchromatography (HPLC) or immunoaffinity chromatography.

In this Example, endothelial cells and fibroblasts are cultured in thepresence of various concentrations (e.g., ng to μg quantities) of therHoxD3 protein or a control recombinant protein produced in the sameexpression system. For these experiments, the use of multiple cell linesis contemplated; including immortalized and primary cells of wild typeand diabetic (db/db) mice. Briefly, the effects of rHoxD3 protein oncollagen synthesis, proliferation and cell viability are examined afterincubation of cultured cells in the presence of rHoxD3. Collagensynthesis is measured by Northern blot as described in Example 5.

To determine the effect of rHoxD3 protein on cell proliferation, therate of DNA synthesis of cultured cells, is quantitated by measuringbromodeoxyuridine (BrdU) incorporation. After incubation of cells for 4or 12 hours with 10 μM BrdU, cells are fixed with 70% ethanol andstained with an anti-BrdU kit (Boehringer), followed by staining with0.5 μg/ml DAPI (4,6 diamidino-2-phenylindole; Sigma). The percentage ofBrdU-positive nuclei is determined by counting multiple fields.

To determine the effect of rHoxD3 protein on cell viability, the use ofany number of techniques is appropriate. For instance, lactatedehydrogenase (LDH) release from cells is measured to assess toxicity ofthe recombinant protein preparations (Korzeniewski and Callewaert, J.Immunol. Methods, 64:313–320 [1983]). Alternatively, apoptosis rates areexamined by annexin V staining or terminaldeoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL)assays, as known in the art (Koopman et al., Blood 84:1415–20 [1994];Gavrieli et al., J. Cell Biol. 119:493–501 [1992]; and Gorczyca et al.,Cancer Res. 53:1945–1951 [1993]).

EXAMPLE 12 Effects of Recombinant HoxD3 Protein on Wounds

For the experiments described in this Example, the transfer ofrecombinant HoxD3 (rHoxD3) protein to wounds of diabetic mice iscontemplated. As the experiments described above indicate thatexpression of HoxD3 is decreased in wounds of diabetic (db/db) animals,experiments are conducted using diabetic (db/db) mice withfull-thickness wounds administered as described in Example 1.Methylcellulose pellets containing various quantities (e.g., 1 ng to 10μg) of rHoxD3 protein are applied directly to the newly made diabeticmouse wounds. Control (diabetic) animals receive methylcellulose pelletscontaining a control recombinant protein. The wounds are measured weeklyuntil closure. It is contemplated that the diabetic wounds treated withthe rHoxD3 protein pellets close in a shorter period of time than thediabetic wounds treated with the control recombinant protein.

In addition, wound biopsies are taken on days 7, 10, 14 and 17, in orderto examine angiogenesis histologically and to assess collagen mRNAexpression levels by Northern blot. Tissue sections are processed forhistochemistry as described in Example 3. The tri-chrome staining methodis used to detect collagen deposition. RNA is isolated and Northern blotanalyses are completed on mouse wound tissue as described in Example 5.Briefly, expression of type I collagen (e.g., Col1A1 mRNA) isquantitated by normalization to rRNA. It is contemplated that the degreeof collagen deposition in diabetic wounds treated with the recombinantHoxD3 protein pellets is greater than that observed in diabetic woundstreated with the control recombinant protein.

EXAMPLE 13 Kinetics of HoxD3 Expression During Wound Healing In Humans

In order to obtain a temporal description of HoxD3 expression during thewound healing process, 4 mm biopsies of intact skin from 6 patients weretaken by hole punch. Additionally, skin biopsies were harvested at 1hour, 1 day, 5 days, and 15 days after wounding. The biopsiescorresponding to a given time point, were pooled. RNA from the biopsiedmaterial was isolated by homogenizing the biopsies in RNAclean buffer(AGS, Heidelberg), to which 1/100 part by volume of 2-mercaptoethanolhad been added. The RNA was then extracted by treatment with acidicphenol saturated with water twice, followed by extraction in thepresence of 1-bromo-3-chloropropane. The RNA was then precipitated withisopropanol and ethanol, and washed with 75% ethanol. After this, aDNase I digestion of the RNA was carried out. For this, 20 μg of RNA (to50 μl with DEPC-treated water) was incubated at 37° C. for 20 min with5.7 μl of transcription buffer (Roche), 1 μl of RNase inhibitor (Roche;40 U/μl) and 1 μl of DNase I (Roche; 10 U/μl). A second digestion stepat 37° C. for 20 min was initiated by addition of 1 μl of DNase I. TheRNA was then treated with phenol, ethanol-precipitated and washed. Allabove mentioned steps were carried out using DEPC (diethylpyrocarbonate)-treated solutions or liquids containing no reactive aminogroups. cDNA was then prepared from the extracted RNA in the presence of1×TaqMan RT buffer (Applied Biosystems), 5.5 mM MgCl₂ (Perkin Elmer),500 μM of each dNTP (Perkin Elmer), 2.5 μM of random hexamers (PerkinElmer), 1.25 U/μl of MultiScribe Reverse Transcriptase (50 U/μl, PerkinElmer), 0.4 U/μl RNase inhibitor (20 U/μl, Perkin Elmer), 20 μl of RNA(50 ng/μl) and DEPC-treated water (to 100 μl volume). After addition ofthe RNA and thorough mixing, the solution was divided in half and placedin 0.2 ml wells (50 μl each) in order to carry out the reversetranscription step in a thermocycler (10 min at 25° C.; 30 min at 48° C.and 5 min at 95° C.). The cDNA was subsequently quantified by means ofquantitative PCR using SYBR green PCR master mixes (Perkin Elmer). Atriplicate determination was made for each cDNA species (in each casewith hoxD3 primers and cyclophilin primers). The stock solution for eachcDNA quantitation contained 37.5 μl of 2×SYBR master mix, 0.75 μl ofAmpErase UNG (1 U/μl) and 18.75 μl of DEPC-treated water (in a totalvolume of 57 μl). Per triplicate determination, 1.5 μl of each primerwere added to 57 μl of stock solution in a previously optimizedconcentration ratio. The HoxD3 primers used included: hoxD3-Primer 1,5′-GCT GCT TAC TAT GAA AAC CCA GG-3′ (SEQ ID NO:5); and hoxD3-Primer2,5′-CGT AAG TGT CCG TAG TTT TGC TGT-3′ (SEQ ID NO:6). Sixty μl of thestock solution/HoxD3 primer solution was mixed with 15 μl of the cDNAsolution (2 ng/μl) and subdivided into 3 reaction wells. Parallel tothis, a stock solution with primers was prepared as a reference for thedetermination of cyclophilin. The cyclophilin primers used included:Cyclophilin-Primer 1, 5′-TCT TAA CCA CCA GAT CAT TCC TTC T-3′ (SEQ IDNO:7); and Cyclophilin-Primer 2, 5′-CCA TAG TGC GAG CAA ATG GG-3′ (SEQID NO:8). Sixty μl of the stock solution/cyclophilin primer solution wasmixed with 15 μl of the same cDNA solution and subdivided into 3reaction wells. Additionally, in order to set up a standard curve forthe Cyclophilin-PCR, various cDNA solutions were prepared as a dilutionseries (4 ng/μl; 2 ng/μl; 1 ng/μl; 0.5 ng/μl and 0.25 ng/μl). Fifteen μlof each of these cDNA solutions were mixed with 60 μl of the stocksolution/cyclophilin primer mixture and subdivided into 3 reaction wellsfor the determination of cyclophilin concentration. A standard curve forhuman HoxD3 was set up in the same manner. As a control, PCR reactionswere run in the absence of cDNA by addition of 15 μl of DEPC water to 60μl of both the stock solution/HoxD3 primer mixture and the stocksolution/Cyclophilin primer mixture. Each solution was then subdividedinto 3 reaction wells prior to thermocycling. The amplification of thebatches was carried out in the GeneAmp 5700 (2 min at 50° C.; 10 min at95° C., followed by 3 cycles of 15 sec at 96° C. and 2 min at 60° C.;then 37 cycles of 15 sec at 95° C. and 1 min at 60° C.). The analysiswas carried out by the determination of the relative abundance of humanHoxD3 with respect to the Cyclophilin reference. For this, a standardcurve was first set up by plotting the C_(T) values of the dilutionseries, against the logarithm of the amount of cDNA in the PCR batch (ngof transcribed RNA) and the slopes (s) of the straight lines wassubsequently determined. The efficiency (E) of the PCR results wascalculated using the following equation:E=10^(−1/s)−1  Equation 1:The relative abundance (X) of human HoxD3 (Y) was then determined inrelation to Cyclophilin (cyc) using the following equation:

${{Equation}\mspace{14mu} 2\text{:}\mspace{14mu} X} = \;\frac{\left( {1 + E_{Cyc}} \right)_{\mspace{14mu} T}^{C\mspace{20mu}{({Cyc})}}}{\left( {1 + E_{Y}} \right)_{\mspace{14mu} T}^{C\mspace{20mu}{(Y)}}}$The numerical values were standardized by setting the amount of HoxD3cDNA obtained from intact skin equal to 1. As is shown in Table 1, whilea downregulation of HoxD3 expression was observed during the earlyphases of wound healing (e.g., 24 hours and 5 days after wounding), anupregulation appears to be necessary for the long-term processes ofhealing (e.g., 15 days after wounding). This demonstrates that theregulation of HoxD3 expression is critical for wound healing and thatdelivery of HoxD3 to wounds according to methods provided by the presentinvention will support wound healing especially in regard to long-termhealing processes.

TABLE 1 HoxD3 Expression During Wound Healing Tissue From HealthyIndividuals Relative Amount of HoxD3 mRNA intact skin 1.0  1 hour afterwounding 1.3 24 hours after wounding 0.6  5 days after wounding 0.7 15days after wounding 2.1

EXAMPLE 14 Dysregulated HoxD3 Expression In Diabetic Human Ulcers

In order to demonstrate, that HoxD3 plays a role not only in normalwound healing but in diabetic wound healing as well, biopsies of 6patients with chronic venous ulcers (ulcera venosum) and biopsies of 4patients with diabetic foot ulcers were taken from intact skin, from thewound ground, and from the wound edge, for analysis of HoxD3 expressionlevels. For each group (intact skin, wound ground, wound edge) thebiopsies of 6 subjects with venous ulcers were pooled. Accordingly,pools were generated for the different biopsy groups from the diabeticulcer patients. RNA was isolated and cDNA was synthesized as describedabove in Example 13. Additionally, HoxD3 cDNA was quantified in relationto Cyclophilin cDNA as previously described, and again the amount ofHoxD3 measured in intact skin was set equal to 1. As shown in Table 2, adysregulation of HoxD3 expression was observed in diabetic ulcers ascompared to normal healing wounds (See Table 1). In particular, whileHoxD3 expression in venous foot ulcer biopsies was only slightlydysregulated as compared to intact skin, a profound lack of HoxD3 mRNAwas observed in biopsies taken from the wounds of the diabetic ulcerpatients.

These results demonstrate that HoxD3 is beneficial for wound healing ingeneral, and moreover, that HoxD3 is especially suitable for treatingdiabetic wounds. Strikingly, during development of the presentinvention, a specific lack of HoxD3 was observed in diabetic wounds butnot in wounds from healthy patients or in badly healing wounds frompatients with venous ulcers.

TABLE 2 HoxD3 Expression In Ulcers HoxD3 mRNA in HoxD3 mRNA in TissueFrom Ulcer Patients Diabetic Foot Ulcers Venous Ulcers intact skin 1.01.0 wound edge 0.2 0.8 wound ground 0.0 1.2

EXAMPLE 15 Cloning and Quantitation of HoxA3 by RT-PCR

Approximately 1 μg of total RNA was reverse transcribed using MMLV RTfor 1 hour at 42° C. in a total volume of 25 μl. Various amounts of thisRT reaction (e.g., 0.1, 1.0, 2.0 and 10.0 μl) were then amplified for20, 30 or 35 cycles of PCR program of 30 sec at 95° C., 30 sec at 58° C.and 90 sec at 72° C. by using the following primers: forward primer5′-TGC GAT CAA GAT CGT GAA ACA ACG C-3′ (SEQ ID NO:17) corresponding tobase pair numbers 93080–93056 of the genomic sequence contained in humanPAC clone (RP-1167F23) given GenBank Accession No:AC004079, and thereverse primer 5′-AGA CTC TCC TGG CGC GTA GCC CCA A-3′(SEQ ID NO:18)corresponding to nucleotides 90277–90312 of the same genomic sequence.The expected 1.37 kb PCR product was visualized by electrophoresis on 1%agarose gels containing ethidium bromide. From this analysis it wasdetermined that amplification of 1 μl of the total 25 μl RT reaction for30 cycles gave optimal, reproducible results within the linear range foramplification. To normalize for total RNA, 1 μl of the same RT reactionwas diluted 1:800 in water and amplified under the same conditions withcommercially available primers sets for human GAPDH or β-actin(Stratagene). The full-length cDNA was cloned by isolating the 1.37 kbHoxA3 PCR product and inserting it into the TOPO II TA cloning vector(Invitrogen). The identity of the insert was confirmed to be HoxA3 byDNA sequencing performed at the UCSF Biomolecular Resource Center.

EXAMPLE 16 Construction of HoxA3 Expression Plasmid

In these experiments, construction of the HoxA3 expression plasmid isdescribed. An EcoR1 fragment from the TOPO II HoxA3 vector describedabove, which lacks the last eight amino acids and stop codon, wasligated in frame into the pCR3.1 myc/his vector (Invitrogen), togenerate a HoxA3 myc/his fusion clone under control of the CMV promoter.A HoxA3 anti-sense vector was prepared using the entire HoxA3 codingsequence ligated into the pCR3.1 vector (Invitrogen) in the anti-senseorientation. Transfections of the HMEC-1 cell line were performed usingthe Effectene reagent (Qiagen) and pools of stably transfected cellswere selected using 35 μg/ml of G418.

EXAMPLE 17 Effects of HoxA3 on Angiogenesis in Chick ChorioallantoicMembranes

To produce replication defective retroviruses encoding HoxA3, the cDNAencoding the entire human HoxA3 sequence was excised with HindIII andPmeI and inserted into the proviral vector CK at the HindIII site and atthe XbaI site, which had been blunted with Klenow polymerase. The viralpackaging cell line Q4dh, derived from the QT6 quail fibrosarcoma cellline (Stoker and Bissell, J. Virol., 62:1008–1015 [1988]) was maintainedin M199 containing 4% FCS, 1% chicken serum and 1× tryptose phosphatebroth. Pools of stable transfectants expressing either a human HoxA3proviral vector or an empty vector (CK) were generated by transfectionusing CaPO₄ and selected in the presence of 200 μg/ml G418 as described(Stoker and Bissell, supra [1988]; and Boudreau et al., J. Cell Biol.,139:257–264 [1997]). To induce angiogenesis, 5×10⁶ transfected Q4dhcells in a volume of 50 μl of M199 medium were grafted onto chickchorioallantoic membranes (CAMs) from 10 day SPAFAS pathogen free chickembryos as previously described (Boudreau et al., supra 1997). After 72hours CAMs were harvested and vascular density, morphology andimmunohistochemical analysis were performed. Angiogenesis wasquantitated by counting the number of branch points arising from thetertiary vessels in a 6 mm square area adjacent to the fibrosarcomatumors. Measurements were made in 12 samples infected with control virusand in 12 samples infected with HoxA3 expressing virus from 3 separateexperiments. Statistical significance was assessed using a pairedt-test.

CAMs were fixed in situ in 4% paraformaldehyde, embedded in OCT medium,frozen in a dry ice/ethanol bath and stored at −70° C. Seven μmcryosections of the CAMs were used for immunohistochemistry. Followingbrief acetone fixation, sections were air-dried and subsequently blockedin PBS containing 2% BSA for one hour followed by staining withappropriate antibodies. A 1:200 dilution of a polyclonalrabbit-anti-human antibody against von Willebrand Factor was usedfollowed by Texas Red conjugated goat anti-rabbit secondary antibody(Calbiochem).

As shown in FIG. 6, significant increase in angiogenesis in the CAM wasobserved following infection with the HoxA3 retrovirus (panels C and D),but not with the control retrovirus (panels A and B). Subsequentsectioning of HoxA3-infected CAM tissues followed by in situhybridization showed high levels of HoxA3 expression in both fibroblastsand endothelial cells adjacent to the virus producing fibrosarcoma cells(See, FIG. 7, panels A and B). Staining of serial sections with a vonWillebrand Factor antibody confirmed an increase in vascular density inthe HoxA3-infected areas (See, FIG. 7, panel C). Furthermore, of over 46HoxA3 retrovirus-infected CAM tissues examined, less than 5% (e.g.,3/46) of the tissues showed evidence of hemorrhagic lesions.

EXAMPLE 18 Gelatin Zymography

Culture media was collected from equal numbers of control HMEC-1, HoxA3or HoxD3 expressing HMEC-1, or from mixed cultures of HoxA3 and HoxD3transfected HMEC-1 cultured in MCDB 131 media containing 5% FCS. Mediawas concentrated two-fold using Centricon 30 microconcentrators(Millipore) and 20 μl of concentrated media was mixed withnon-denaturing sample loading buffer. Proteins were separated by 10%SDS-PAGE containing 0.1% gelatin (BioRad). Following electrophoresis,gels were washed for 60 minutes in 2.5% Triton X-100 in 50 mM Tris HCl,pH 7.6, and developed for 24 hours at 37° C. in 50 mM Tris HCl, pH 7.6,containing 5 mM CaCl₂, 200 mM NaCl and 0.02% Brij-35. The gels werestained with 0.25% Coomassie Blue in 50% methanol and 10% acetic acid.Gels were photographed using an Alpha Innotech Chemlmager 4000 systemequipped with a CCD camera and densitometric software.

The majority of MMP-2, secreted by either control, HoxD3 or HoxA3overexpressing cells migrated at approximately 70 kD corresponding withthe latent form of this protease. However, when equal numbers of HoxA3and HoxD3 overexpressing cells were co-cultured, an additional clearband at approximately 65 kD corresponding to the activated form of MMP-2was observed (See, FIG. 8, panel D).

EXAMPLE 19 DNA Microarray Analysis

A Human Cell-Cell Interaction Array was purchased from Clontech.Approximately 60 μg of total RNA isolated from control or HoxA3transfected HMEC-1 cells was labeled using the Atlas Pure Total RNALabeling System (Clontech). Poly A⁺ RNA enrichment was done using astreptavidin magnetic bead preparation. cDNA probes were synthesizedfollowing reverse transcription and amplification with the suppliedprimer mix and α³²P dATP. Membranes were hybridized according tomanufacturers instructions. Following hybridization, membranes wereexposed to BiomaxMS film (Kodak) for 1–4 days at −70° C. Quantificationof signals was performed by scanning-densitometry and subsequentanalysis was done using NIH Image 1.61 software. Microarray analysis wasperformed on at least two independent samples of RNA harvested fromeither HoxA3 or control transfected cells.

Densitometric analysis revealed at least a two-fold upregulation of anumber of genes in HoxA3-expressing EC in comparison tocontrol-transfected cells. Of note was the increased expression ofMMP-14 (MT-1MMP), the cell surface receptor for the serine proteinaseuPA (uPAR), as well as tenascin, cdc42GTPase and alpha 3 integrin, eachof which have been implicated in promoting angiogenesis or cellmigration (Hiroaka et al., Cell, 95:365–377 [1998]; Kroon et al., Am. J.Pathol., 154:1731–1742 [1999]; Ridley, J. Cell Biol., 150:107–109[2000]; Schenk et al., Mol. Biol. Cell, 10:2933–2943 [1999]; andGonzales et al., Mol. Biol. Cell, 10:259–270 [1997]).

Northern Blot analyses were subsequently performed as described above inExample 5, to provide independent confirmation that the mRNAscorresponding to uPAR and MMP-14 were upregulated in HMEC-1 transfectedwith HoxA3, as compared to control transfected EC (See, FIG. 8, panelA). The expression of uPAR and MMP-14 in HoxD3 transfected HMEC-1 wasalso examined. Interestingly, uPAR and MMP-14 expression was notsignificantly upregulated in HoxD3-transfected cells, underscoring thedistinct phenotypes induced by expression of each of these Hox genes.Western blot analysis of control or HoxA3-transfected HMEC-1 lysates,confirmed that the HoxA3-induced increases in mRNA for both uPAR andMMP-14 were accompanied by increased expression of the respectiveproteins (See, FIG. 8, panel B). Because MMP-14 acts primarily toactivate latent MMP-2, the expression of MMP-2 in HoxA3 andHoxD3-transfected EC was examined. While levels of MMP-2 RNA wereunchanged in HoxA transfected cells, overexpression of the paralogousHoxD3 gene, strongly upregulated expression of MMP-2 (See, FIG. 8, panelC). Together these results indicate that HoxA3 and HoxD3 genes actcooperatively in EC to regulate both metallo- and serine proteinaseactivity during angiogenesis.

EXAMPLE 20 Fibrin and Fibrinogen Migration Assays

In this example, the methods used to assess the effects of HoxA3 andHoxD3 on cell migration in three-dimensional fibrin matrices aredescribed. Specifically, migration of control HMEC-1 or HMEC-Itransfected with a HoxA3 antisense construct was examined. Five hundredμl of a solution containing 1 mg/ml Cytodex-3 gelatin coatedmicrocarrier beads (Pharmacia) in PBS were seeded with 5×10⁵ HMEC-1cells and maintained in suspension culture in bacterial plates in MCDB131 media containing 10% FCS until the cells reached confluency. Thecells cultured with microcarriers were subsequently embedded intothree-dimensional fibrin gels prepared as described (Nehls andDrenckhahn, Microvasc. Res., 50:311–322 [1995]). Briefly, 0.5 ml ofbeads/HMEC-1 cells, were suspended in a mixture containing 800 μl of a5.45 mg/ml fibrinogen solution (in PBS, pH 7.2) and 300 μl of a 2 U/mlthrombin solution (both obtained from Sigma) and incubated at 37° C. for30 minutes to allow fibrin clotting. At the indicated times, 50 ng ofrecombinant human bFGF (R&D Systems) or 25 μg/ml of control IgG orfunction blocking antibodies were also included in the gel mixture. Thefunction blocking antibodies tested included antibodies against humanuPAR (#399R from American Diagnostica), αvβ3 integrin (LM609 from D.Cheresh of Scripps), and GM6001 (Chemicon International). Following gelclotting, 1 ml of MCDB 131 media containing 5% FCS was added. Freshmedia (with or without soluble factors) was added to the fibrin matricesat 48 hour intervals. Cell migration was observed by phase contrastmicroscopy using a Nikon TE300 inverted microscope and photographedusing a Hamamatsu Orca digital camera and Open Lab Improvision software.Migration on fibrinogen substrates was measured as previously described(Myers et al., J. Cell Biol., 148:343–352 [2000]).

Whereas unstimulated control transfected HMEC-1 display a limitedability to invade the fibrin gels, addition of bFGF enhanced migration,which in turn was completely blocked by addition of function blockingantibodies against uPAR (See, FIG. 9, panel A). Importantly, even in thepresence of bFGF, HMEC-1 transfected with a HoxA3 antisense constructfailed to invade the fibrin gels.

In contrast, overexpression of HoxA3 in HMEC-1 induced extensivespontaneous invasion into fibrin gels even in the absence of exogenousbFGF (See, FIG. 9, panel B). The HoxA3-induced migration couldsubsequently be attenuated by addition of anti-uPAR antibodies. Theaddition of the metalloproteinase inhibitor GM6001, had no significanteffect on either bFGF or HoxA3-stimulated migration in this system.HoxD3-transfected HMEC-1 also spontaneously migrated into the fibringels with out addition of bFGF, but to a lesser degree than thatobserved for HoxA3-transfected HMEC-1 (See, FIG. 9, panel B).HoxD3-induced migration was also attenuated by addition of anti-uPARantibodies.

The ability of HoxD3 and HoxA3-transfected EC to migrate on a fibrinogensubstratum was then compared. Although HoxA3-transfected EC displayed anenhanced ability to migrate as compared to control transfected EC, thismigration was significantly less than migration induced by transfectionof HoxD3 (See, FIG. 9, panel C). Together these results indicate thatHoxA3 and HoxD3 selectively facilitate EC migration in differentmicroenvironments.

EXAMPLE 21 Modulation of HoxA3 Expression by EC Microenvironment

HMEC-1 was cultured on tissue culture plastic, or on top of thick layersof reconstituted basement membranes (e.g., Matrigel), where cellsundergo morphological reorganization into tube-like structures andbecome growth arrested within 24 hours (Kubota et al., J. Cell Biol.,107:1589–98 [1988]; and Boudreau et al., J. Cell Biol., 139:257–264[1997]). As shown in FIG. 10, panel A, whereas Hox A3 was expressed atrelatively low levels by in EC made quiescent by culturing on basementmembranes (BM), expression was significantly higher in activated ECcultured on tissue culture plastic. Treatment of quiescent EC with 30ng/ml bFGF for 18 hours also increased expression of HoxA3 (See, FIG.10, panel B).

EXAMPLE 22 HoxA3 Expression During Wound-Induced Angiogenesis In Vivo

C57BL mice obtained from Jackson Laboratories were anaesthetized withketamine/xylazine and a 1 cm linear full thickness wound was madethrough the skin. Tissues were harvested after 4 or 7 days, fixed informalin and embedded in paraffin. Seven μm sections were prepared anddeparaffinized by heating at 80° C. for 30 minutes followed by twowashes in xylenes for 5 min as described (Uyeno et al., J. Surg. Res.,100:46–56 [2001]). Sections were rehydrated through an ethanol series,post-fixed for 5 minutes with 4% paraformaldehyde, digested with 1 μg/mlProteinase K (Sigma) for 10 minutes and hybridized using 800 ng/ml ofdigoxigenin-labelled riboprobes as described (Boudreau et al., J. CellBiol., 139:257–264 [1997]). Riboprobes against HoxA3 were generatedusing a RNA Dig labelling kit (Boehringer) with either T7 or Sp6 RNApolymerase from a 395 bp KpnI/EcoRI fragment of the 3′ end of humanHoxA3 subcloned into the TOPO II PCR cloning vector (See, Example 15).

Hox A3 expression in angiogenic or quiescent endothelial cells in vivowas assessed by in situ hybridization on control or wounded dermaltissue. While levels of HoxA3 were observed to be low in resting vesselsin unwounded skin (See, FIG. 10, panel C), HoxA3 was upregulated in EC 4days following administration of a full thickness linear wound (See,FIG. 10, panel D). These results indicate that like HoxD3, HoxA3 isupregulated in angiogenic environments and also contributes toangiogenesis during wound repair.

EXAMPLE 23 Effects of HoxA3 DNA Application on Wound Closure In Vivo

Genetically diabetic (db/db) mice were anesthetized with 0.04 cc of aketamine:xylazine mixture (50 mg/cc:2.5 mg/cc), and the dorsum of themouse was shaved prior to creation of a 2.5 cm wound by excision of thepanniculus camosus layer. Immediately following wounding 4 pelletsconsisting of a mixture of 1% methylcellulose and 25 μg of either PgalcDNA or HoxA3 cDNA were applied to the wounds. The diameter of thewounds was measured immediately following wounding and then every 7 daysuntil the wounds were completely healed. The NIH Image J analyzer wasused to determine the area of the wound tracing, the size of which wascompared by student's t test.

Strikingly, within 14 days, the HoxA3 cDNA treated wounds appearedsmaller than those treated with control cDNA (See, FIG. 11, panels A andB). Application of HoxA3 also significantly improved the rate of woundclosure, with HoxA3-treated wounds closing at approximately 28–35 days,as compared to wounds treated with control DNA which take 77 days toclose (See, FIG. 11, panel C). An increase in the number of bloodvessels present in the HoxA3 treated mice, as compared to controltreated animals was also observed. Thus although both HoxA3 and HoxD3can induce angiogenesis and wound closure, HoxA3 was even more effectivethan HoxD3 in accelerating wound closure.

EXAMPLE 24 Effects of HoxA3 Expression on Wound Closure In Vitro

This example describes methods suitable for assessment of the effects ofHoxA3 expression on closure of a scratch wound in a tissue cultureassay. The cDNA encoding the human HoxA3/myc-his fusion protein iscloned into the retroviral vector, pLXSN (Clontech). The MK line ofimmortalized mouse keratinocytes (obtained from D. Morris of UCSF) arethen infected with either a HoxA3 retrovirus or a PLXSN/GFP retrovirus.Stable colonies are selected using G418. Cultures of keratinocytesexpressing HoxA3 or GFP are then grown to confluence and scratch woundsare administered using a 1 cc syringe. Cells are examined andphotographed at 24, 48 and 72 hours following wounding. The number ofcells migrating into the wounded area (e.g., scratched), are thencounted and compared using a student's t test. In addition, RNA isharvested from the cells, 72 hours following administration of thescratch wounds. RT-PCR and Northern blot analysis are performed usingthe RNA from the HoxA3 and GFP expressing keratinocytes.

The expression of HoxA3 is contemplated to result in an increased numberof keratinocytes migrating into the wound area as compared to controlcells. As shown in FIG. 13, panels A–D, a significantly greater numberof HoxA3+ cells migrated into the wound area as compared to both thecontrol cells and HoxD3+ cells. Additionally, HoxA3 retrovirus infectedkeratinocytes are contemplated to express greater amounts of alpha 3integrin (as was observed in endothelial cells which overexpress HoxA3).

EXAMPLE 25 Cloning and Expression of HoxB3

Approximately 1 μg of total RNA was reverse transcribed using MMLV RTfor 1 hour at 42° C. in a total volume of 25 μl. Various amounts of thisRT reaction (e.g., 0.1, 1.0, 2.0 and 10.0 μl) were then amplified for20, 30 or 35 cycles of PCR program of 30 sec at 95° C., 30 sec at 58° C.and 90 sec at 72° C. by using the following primers: forward primer5′-CGATGCAGAA AGCCACCTAC TACGAC-3′ (SEQ ID NO:19) corresponding to basepair numbers 362–387 of human HoxB3; and reverse primer 5′-CGCCGACCCCGGGGGGCTCT TCT-3′ (SEQ ID NO:20) corresponding to nucleotides1,666–1,682 of the published sequence. The expected 1.32 kb PCR productwas visualized by electrophoresis on 1% agarose gels containing ethidiumbromide. From this analysis it was determined that amplification of 1 μlof the total 25 μl RT reaction for 30 cycles gave optimal, reproducibleresults within the linear range for amplification. To normalize fortotal RNA, 1 μl of the same RT reaction was diluted 1:800 in water andamplified under the same conditions with commercially available primerssets for human GAPDH or β-actin (Stratagene). The 1.32 kb HoxB3 PCRproduct was subsequently ligated into the TOPO II TA cloning vector(Invitrogen), and the identity confirmed by dideoxy sequencing.

The HoxB3 insert containing the entire cDNA encoding human HoxB3 wassubcloned into the expression vector PCR3.1 (Invitrogen) in both senseand antisense directions. The orientation was confirmed by restrictiondigestion. High levels of translation and expression of HoxB3 wasachieved by introduction of a Kozak consensus sequence on the 5′ end byre-amplifying the cDNA with the primer 5′-GGAATTCGGC CACCATGCAG A-3′(SEQ ID NO:21). The resulting cDNA was religated into the PCR3.1 vector.HoxB3 transgene expression was distinguished from endogenous HoxB3expression via a C-terminal His tag introduced by deletion of the HoxB3stop codon, and subcloning into the PCR3.1mycHis vector. Sense andantisense HoxB3 clones were transfected into the HMEC-1 cell line byusing a calcium phosphate method, and stable transfectants weresubsequently selected in the presence of 50 μg/ml of G418.

EXAMPLE 26 Effects of HoxB3 DNA Application on Wound Closure In Vivo

Genetically diabetic (db/db) mice were anesthetized with 0.04 cc of aketamine:xylazine mixture (50 mg/cc:2.5 mg/cc), and the dorsum of themouse was shaved prior to creation of a 2.5 cm wound by excision of thepanniculus camosus layer. Immediately following wounding 4 pelletsconsisting of a mixture of 1% methylcellulose and 25 μg of either βgalcDNA or HoxB3 cDNA were applied to the wounds. The diameter of thewounds was measured immediately following wounding and then every 7 daysuntil the wounds were completely healed. The NIH Image J analyzer wasused to determine the area of the wound tracing, the size of which wascompared by student's t test.

Strikingly, within one week, the HoxB3 cDNA treated wounds appearedsmaller than those treated with control cDNA. Application of HoxB3 alsosignificantly improved the rate of wound closure, with HoxB3-treatedwounds closing at approximately 28 days, as compared to wounds treatedwith control DNA which take 77 days to close (See, FIG. 14).

In summary, the present invention provides numerous advances andadvantages over the prior art, including methods and compositions forthe improvement of wound healing. All publications and patents mentionedin the above specification are herein incorporated by reference. Variousmodifications and variations of the described method and system of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific preferred embodiments, itshould be understood that the invention as claimed should not be undulylimited to such specific embodiments. Indeed, various modifications ofthe described modes for carrying out the invention which are obvious tothose skilled in diagnostics, cell culture, and/or related fields areintended to be within the scope of the present invention.

1. A method comprising: a) providing; i) a subject with a diabetic skinwound, and ii) a composition comprising a gene delivery vehicle and anexpression vector comprising HoxB3 nucleic acid encoding a HoxB3protein, wherein said HoxB3 protein is the protein set forth in SEQ IDNO:23 or a biologically active variant thereof that differs from SEQ IDNO:23 by less than 1%; and b) applying said composition to said woundunder conditions suitable for transfecting at least one cell of saidwound with said expression vector.
 2. The method of claim 1, whereinsaid HoxB3 protein is the protein set forth in SEQ ID NO:23.
 3. Themethod of claim 1, wherein said applying is under conditions such thatwound closure is accelerated.
 4. The method of claim 1, wherein saidapplying is under conditions such that angiogenesis in said wound isenhanced.
 5. The method of claim 1, wherein said composition furthercomprises a cellulosic material.
 6. The method of claim 1, wherein saidwound has a reduced level of expression of HoxD3.
 7. The method of claim1, wherein said expression vector further comprises one or both of HoxD3nucleic acid encoding a HoxD3 protein and HoxA3 nucleic acid encoding aHoxA3 protein, wherein said HoxD3 protein is the protein set forth inSEQ ID NO:2 or a biologically active variant thereof that differs fromSEQ ID NO:2 by less than 1%, and said HoxA3 protein is the protein setforth in SEQ ID NO:15 or a biologically active variant thereof thatdiffers from SEQ ID NO: 15 by less than 1%.
 8. The method of claim 1,wherein said HoxB3 nucleic acid comprises a nucleic acid set forth inSEQ ID NO:22.
 9. The method of claim 1, wherein said composition islocated in a wound care device.
 10. The method of claim 1, wherein saidwound is an ulcer.
 11. The method of claim 7, wherein said HoxD3 proteinis the protein set forth in SEQ ID NO:2.
 12. The method of claim 7,wherein said HoxD3 nucleic acid comprises the nucleic acid set forth inSEQ ID NO:1.
 13. The method of claim 7, wherein said HoxA3 protein isthe protein set forth in SEQ ID NO:15.
 14. The method of claim 7,wherein said HoxA3 nucleic acid comprises the nucleic acid set forth inSEQ ID NO:14.
 15. The method of claim 1, wherein said gene deliveryvehicle is selected from the group consisting of gel matrices,liposomes, virosomes, cationic lipids, polylysine, adenoviral vectors,retroviral vectors and gold particles.
 16. A method comprising: a)providing; i) a subject with a diabetic skin wound, and ii) acomposition comprising a cellulosic material and an expression vectorcomprising HoxB3 nucleic acid encoding the HoxB3 protein set forth inSEQ ID NO:23; and b) applying said composition to said wound underconditions suitable for transfecting at least one cell of said woundwith said expression vector.
 17. The method of claim 16, wherein saidcellulosic material comprises methyl cellulose pellets comprising saidexpression vector.
 18. The method of claim 17, wherein said HoxA3nucleic acid comprises the nucleic acid set forth in SEQ ID NO:22.